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IMPROVING THE THERMOSTABILITY AND INCREASING THE SUBSTRATE RANGE OF OLD YELLOW ENZYME HOMOLOGS AND AMINOLEVULINIC ACID SYNTHASE
THROUGH PROTEIN ENGINEERING
By
ROBERT WILSON POWELL III
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2016
© 2016 Robert Wilson Powell III
“To my family and friends”
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ACKNOWLEDGMENTS
I would like to acknowledge my faculty advisor Dr. Jon Stewart for his guidance. I
am very grateful to him for the opportunity to do work in his lab. I feel fortunate to have
been able to work on projects which I enjoyed so much. I am very proud of the work we
do in this lab and am grateful to have been able to contribute what I could during my
time. I would like to acknowledge the University of Florida. And, I would like to
acknowledge the NSF for funding my research.
I would like to acknowledge Dr. Bradford Sullivan for his assistance during the
OYE 1 Y196 project. I would like to acknowledge Athena Patterson for her assistance
during the OYE2.6 Y78Xsm / I113Xsm project. I would like to acknowledge Steven
Crichton for all his work during the OYE2.6 Y78Xsm / I113Xsm and OYE2.6 Y78Xsm /
I113C / F247X projects. I would like to acknowledge Dr. Filip Boratynski for all of his
work during the OYE 2.6 thermostability project. I would like to acknowledge Matthew
Burg and Dr. Steven Bruner for all of their help during the OYE 3 crystallization project. I
would like to acknowledge Steven Crichton again for all his work during the mALAS
project. Every one of these people have contributed to these projects in a meaningful
way and I am grateful to them all for it.
I would like to thank Dr. Hillary Lathrop for all her help with the Beckman and
Gilson HPLCs as well as for all her work maintaining our instrument room. I would like
to acknowledge Sarah Franz for all her help with the MSTFA derivatization protocols. I
would like to thank Louis Mouterde for all his work with the acyl-CoA separation method
development. I am grateful for their help running and maintaining the lab. I would like to
thank my family and friends for their support. I would especially like to thank my mom,
Janet Powell, for all of her support during my time here. And, I would also like to thank
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Nicole Gibbons for her love and support. I am very thankful for all the help Nicole has
given me during our time here. I am very grateful to have met her and to have been able
to go on this journey with her.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES .......................................................................................................... 11
LIST OF FIGURES ........................................................................................................ 12
LIST OF ABBREVIATIONS ........................................................................................... 19
ABSTRACT ................................................................................................................... 20
CHAPTER
1 PROBING POSITION Y196 IN OLD YELLOW ENZYME 1 .................................... 22
Background ............................................................................................................. 22
Isolation and Purification of OYE ...................................................................... 22 Catalytic cycle and substrates of OYE ............................................................. 23 Structure and Mechanism of OYE .................................................................... 24
Project Overview .............................................................................................. 25 Results and Discussion........................................................................................... 25
OYE 1 YI96 Site-Saturation Mutagenesis Library ............................................. 25 Crystal Structure of OYE 1 YI96C .................................................................... 27
Experimental ........................................................................................................... 28 General............................................................................................................. 28 Cloning ............................................................................................................. 29
Construction of plasmids used to make the OYE 1 Y196 library ................ 29 Construction of mutants in the OYE 1 Y196X randomized library .............. 29
Substrates ........................................................................................................ 30 2-(Hydroxymethyl)-cyclopent-2-enone (1) .................................................. 30 2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................... 31
Methyl 2-(hydroxymethyl)acrylate (3) ......................................................... 31 (S)-(+)-Carvone (7) .................................................................................... 31
(R)-(-)-Carvone (8) ..................................................................................... 32
2-Methyl-2-cyclopenten-1-one (11) ............................................................ 32
2-Methyl-2-cyclohexen-1-one (12) ............................................................. 32 Screening Assay .............................................................................................. 33 Protein Purification and Crystallogenesis of Y196C OYE 1 .............................. 33 The Data Collection and Crystal Structure of OYE 1 Y196 Mutants ................. 35
Conclusions ............................................................................................................ 36
2 IMPROVING THE PRODUCT RANGE OF OYE 1 AND OYE 2.6 THROUGH PROTEIN ENGINEERING ...................................................................................... 61
Background ............................................................................................................. 61
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Mutagenesis of the East Side of the Active Site in OYE ................................... 61
Project Summary .............................................................................................. 64 Results and Discussion........................................................................................... 65
OYE 2.6 Y78Xsm / I113Xsm Randomized Library .............................................. 65 OYE 2.6 Y78Xsm / I113Xsm Presequenced Library ........................................... 68 OYE 2.6 Y78Xsm / I113C / F247X Randomized Libraries ................................. 68
Experimental ........................................................................................................... 70 General............................................................................................................. 70
Cloning ............................................................................................................. 70 Construction of plasmids used to make the OYE 2.6 ................................. 70 Construction of the mutants in the OYE 2.6 Y78Xsm / I113Xsm
randomized library .................................................................................. 70 Construction of the mutants in the OYE 2.6 Y78Xsm-I113Xsm
presequenced library .............................................................................. 73 Addressing concatomeric primer inserts .................................................... 74
Construction of mutants in the OYE 2.6 Y78Xsm / I113C / F247X randomized libraries ............................................................................... 74
Substrates ........................................................................................................ 75 Methyl 2-(hydroxymethyl)acrylate (1) ......................................................... 75
2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................... 76 2-(Hydroxymethyl)-cyclopent-2-enone (3) .................................................. 76
Screening Assay .............................................................................................. 76 Conclusions ............................................................................................................ 77
3 IMPROVING THE THERMOSTABILITY OF OYE 2.6 THROUGH PROTEIN ENGINEERING ....................................................................................................... 95
Background ............................................................................................................. 95
Improving Thermostability through Mutagenesis .............................................. 95 Project Summary .............................................................................................. 97
Results and Discussion........................................................................................... 98 Residues with High Local B-Factors ................................................................. 98 Dimer Interface Residues ............................................................................... 100
Combining Thermostabilizing Mutations ......................................................... 100 Crystallization of OYE 2.6 D141E-S388P ....................................................... 101
Experimental ......................................................................................................... 104 General........................................................................................................... 104
Cloning ........................................................................................................... 104 Construction of the plasmid used as a template for the OYE 2.6 thermal
stability libraries .................................................................................... 104 Construction of OYE 2.6 libraries ............................................................. 105
Substrates ...................................................................................................... 106
2-Methyl-2-cyclopenten-1-one (1) ............................................................ 106 Screening ................................................................................................. 107
The Protein Purification and Crystallization of OYE 2.6 Mutants .................... 109 Data Collection and Structure Solution of OYE 2.6 D141E-S388P ................ 112 B-Factor Data and Statistics ........................................................................... 113
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Conclusions .......................................................................................................... 113
4 THE STRUCTURE OF Saccharomyces cerevisiae OLD YELLOW ENZYME 3 ... 135
Background ........................................................................................................... 135
Crystallization of OYE Family Members ......................................................... 135 Project Summary ............................................................................................ 136
Results and Discussion......................................................................................... 137 Crystallization of OYE 3.................................................................................. 137 Data Reduction and Structure Solution .......................................................... 139
OYE 3 W116 Site Saturation Mutagenesis ..................................................... 143 X-Ray Crystallography Studies of OYE 3 W116 Mutants and Related
Proteins ....................................................................................................... 144
Experimental ......................................................................................................... 146 General........................................................................................................... 146 Cloning ........................................................................................................... 146
Construction of plasmid used for libraries ................................................ 146 Construction of an OYE 3 W116 site-saturation mutagenesis library ....... 147
Testing of Phenol Binding to OYE 3 ............................................................... 149 GST-OYE 3 Fusion Protein Purification and Crystallogenesis ....................... 149 Native OYE 3 Protein Purification and Crystallogenesis ................................ 150
Alkene Substrate for OYE 3 ........................................................................... 154 2-(Hydroxymethyl)-cyclopent-2-enone (1) ................................................ 154
2-(Hydroxymethyl)-cyclohex-2-enone (2) ................................................. 154 (R)-Pulegone (3) ...................................................................................... 155 (S)-(+)-Carvone (4) .................................................................................. 155 (R)-(-)-Carvone (5) ................................................................................... 156
2-Methyl-2-cyclopenten-1-one (11) .......................................................... 156
2-Methyl-2-cyclohexen-1-one (12) ........................................................... 156 3-Methyl-cyclohexen-1-one (13) .............................................................. 157
3-Ethyl-cyclohexen-1-one (14) ................................................................. 157 3-Methyl-cyclopenten-1-one (15) ............................................................. 157 4-Ethyl-4-methyl-2-cyclohexen-1-one (21) ............................................... 158
4-Isoproply-4-methyl-2-cyclohexen-1-one (22) ........................................ 158 4,4-Diethyl-2-cyclohexen-1-one (23) ........................................................ 158 Spiro[5.5]undec-1-en-3-one (24) .............................................................. 159 2-Butylidenecyclohexanone (25) .............................................................. 159
Screening ....................................................................................................... 159 Conclusions .......................................................................................................... 160
5 IMPROVING THE SUBSTRATE RANGE OF AMINOLEVULINIC ACID SYNTHASE THROUGH PROTEIN ENGINEERING............................................. 190
Background ........................................................................................................... 190
Positions of Interest ........................................................................................ 191 Threonine 148 .......................................................................................... 191
Isoleucine 151 .......................................................................................... 191
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Arginine 85 ............................................................................................... 192
The Glycine Loop ........................................................................................... 192 Project Overview ............................................................................................ 193
Results and Discussion......................................................................................... 195 Detecting δ-AL-pyrrole Compounds with Ehrlich’s Reagent ........................... 195 Preparation and Detection of Succinyl-CoA ................................................... 196 In Situ Succinyl-CoA Formation ...................................................................... 196
Detecting Amino Products Using PITC and MSTFA Derivatives .................... 197
mALAS R85, T148 and I151 Site-Saturation Mutagenesis Libraries .............. 199 Experimental ......................................................................................................... 199
General........................................................................................................... 199 Cloning ........................................................................................................... 200
Construction of plasmid used to make ALAS libraries ............................. 200
Construction of ALAS libraries ................................................................. 200 Preparation and Detection of Succinyl-CoA ................................................... 202
Succinyl-CoA Regeneration System .............................................................. 203
Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent .......................... 204 Derivatizing Amino Acids Using PITC ............................................................ 205 Detecting PITC Amino Acid Derivatives by HPLC .......................................... 206
Derivatizing Amino Acids Using MSTFA......................................................... 206 Detecting MSTFA Amino Acid Derivatives Using GC-MS .............................. 207
Conclusions .......................................................................................................... 207
APPENDIX
A LIST OF PRIMERS ............................................................................................... 223
Chapter 1 Primers ................................................................................................. 223 Chapter 2 Primers ................................................................................................. 224
Chapter 3 Primers ................................................................................................. 225 Chapter 4 Primers ................................................................................................. 226
Chapter 5 Primers ................................................................................................. 228
B MUTAGENIC PLASMIDS ..................................................................................... 232
C PLASMID SEQUENCES ....................................................................................... 233
Sequence of pET3b-OYE1 ................................................................................... 233
Sequence of pBS2 ................................................................................................ 235 Sequence of pFB1 ................................................................................................ 238 Sequence of pRP4 ................................................................................................ 241
Sequence of pGF23 .............................................................................................. 243
D GC AND HPLC METHODS .................................................................................. 246
AZW2.Meth ........................................................................................................... 246 AZW3.Meth ........................................................................................................... 246 BTS2.Meth ............................................................................................................ 247
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BTS3.Meth ............................................................................................................ 247
BTS4.Meth ............................................................................................................ 248 BTS7.Meth ............................................................................................................ 248
BTS8.Meth ............................................................................................................ 249 FB1.Meth .............................................................................................................. 249 JON.Meth .............................................................................................................. 250 SEF.Meth .............................................................................................................. 250 YAP.Meth .............................................................................................................. 251
LMM.Meth ............................................................................................................. 251 RWP2.Meth .......................................................................................................... 252
E PLASMID MAPS ................................................................................................... 253
pET3b-OYE .......................................................................................................... 253 pBS2 ..................................................................................................................... 253 pFB1 ..................................................................................................................... 254
pRP4 ..................................................................................................................... 254 pGF23 ................................................................................................................... 255
LIST OF REFERENCES ............................................................................................. 256
BIOGRAPHICAL SKETCH .......................................................................................... 263
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LIST OF TABLES
Table page 2-1 List of the presequenced alkene reductase libraries ........................................... 79
2-2 OYE 2.6 best variants discovered during the OYE 2.6 ISM studies ................... 80
2-3 Q scores for NNK randomized libraries. ............................................................. 80
3-1 Crystallographic data collection and refinement statistics ................................ 116
3-2 Q scores for NNK randomized libraries. ........................................................... 117
4-1 Crystallographic data collection and refinement statistics. ............................... 162
4-2 Crystallographic data collection and refinement statistics. ............................... 163
4-3 Distances between the β-carbon of each active site residue to the ligand ....... 164
5-1 Retention times of PIT-amino acid derivative standards from HPLC ................ 209
A-1 List of mutagenic primers for chapter 1. ........................................................... 223
A-2 List of mutagenic and sequencing primers for chapter 2 .................................. 224
A-3 List of mutagenic and sequencing primers for chapter 3 .................................. 225
A-4 List of mutagenic and sequencing primers for chapter 4 .................................. 226
A-5 List of mutagenic and sequencing primers for chapter 5 .................................. 228
B-1 Mutagenic plasmids used in this study. ............................................................ 232
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LIST OF FIGURES
Figure page 1-1 The catalytic cycle of G6PDH investigated by Warburg and Christian ............... 38
1-2 The catalytic cycle of OYE 1 established by Massey and Vas ........................... 38
1-3 List of OYE 1 substrates from the literature ........................................................ 39
1-4 FMN Diagram displaying the FMN environment in the active site of OYE 1 ....... 50
1-5 Catalytic mechanism of OYE 1 ........................................................................... 50
1-6 OYE substrate binding modes ............................................................................ 51
1-7 OYE 1 active site diagram .................................................................................. 52
1-8 List of Baylis-Hillman substrates screened by OYE 1 Y196 library ..................... 53
1-9 List of carvone substrates screened by OYE 1 Y196 library ............................... 54
1-10 List of screening substrates screened by OYE 1 Y196 library ............................ 55
1-11 Calculations for obtaining a Qscore of a pooled plasmid mix from a NNK primer mix using data from a theoretical sequencing electropherogram ....................... 56
1-12 OYE 1 Y196 library screening results for 1 ......................................................... 57
1-13 OYE 1 Y196 library screening results for 2 ......................................................... 57
1-14 OYE 1 Y196 library screening results for 3 ......................................................... 58
1-15 OYE 1 Y196 library screening results for 7 ......................................................... 58
1-16 OYE 1 Y196 library screening results for 8 ......................................................... 59
1-17 OYE 1 Y196 library screening results for 11 ....................................................... 59
1-18 OYE 1 Y196 library screening results for 12 ....................................................... 60
1-19 Alignment of OYE 1 wt. and OYE 1 Y196C ........................................................ 60
2-1 Flipped binding mode ......................................................................................... 81
2-2 List of Chapter 2 substrates ................................................................................ 82
2-3 (S)-(+)-carvone bound in a flipped binding mode to the active site of OYE 1 W116I ................................................................................................................. 83
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2-4 Mechanism of OYE 2.6 ....................................................................................... 83
2-5 Diagram of the residues in the OYE 2.6 active site ............................................ 84
2-6 Malonate bound in the active site of OYE 2.6 Y78W-I113C ............................... 84
2-7 Substrate 1 and 2 modeled into the active of OYE 2.6 Y78W and OYE 2.6 wt .. 85
2-8 Sequence alignment of OYE 2.6 with a sample containing primer inserts .......... 85
2-9 Best variants discovered during both the ISM project and this project using a small residue matrix for obtaining the (R)-5 product from substrate 2 ................ 86
2-10 OYE 2.6 Y78Xsm / I113Xsm library screening results for substrate 2 ................ 87
2-11 OYE 2.6 Y78A / I113C / F247X screening results for substrate 2 ...................... 88
2-12 OYE 2.6 Y78C / I113C / F247X screening results for substrate 2 ...................... 89
2-13 OYE 2.6 Y78G / I113C / F247X screening results for substrate 2 ...................... 90
2-14 OYE 2.6 Y78S / I113C / F247X screening results for substrate 2 ...................... 91
2-15 OYE 2.6 Y78T / I113C / F247X screening results for substrate 2....................... 92
2-16 OYE 2.6 Y78V / I113C / F247X screening results for substrate 2 ...................... 93
2-17 Calculations for obtaining a Qscore of a pooled plasmid mix from a KST primer mix using data from a theoretical sequencing electropherogram ....................... 94
3-1 The fraction of B-factors for each position over the average B-factors of all positions in the structure ................................................................................... 118
3-2 The relative B-factors of all three published OYE 2.6 wild type structures ....... 118
3-3 The positions targeted during the ISM thermostability project, their region in the protein, and their B-factors for structure 3TJL ............................................ 119
3-4 The positions targeted during the local maximum project, their region in the protein, and their B-factors for structure 3TJL .................................................. 119
3-5 The B-factor values for the positions targeted during the local maximum project ............................................................................................................... 120
3-6 The positions selected for mutagenesis during the dimer interface project ...... 120
3-7 The results for the small scale screening of the OYE 2.6 S388 library testing all 19 possible replacements with substrate 1 .................................................. 121
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3-8 The results of screening the OYE 2.6 E41X NNK randomized library .............. 121
3-9 The results of screening the OYE 2.6 D141X NNK randomized library ............ 122
3-10 The results of screening the OYE 2.6 E145X NNK randomized library ............ 122
3-11 The results of screening the OYE 2.6 K330X NNK randomized library ............ 123
3-12 The results of screening the OYE 2.6 I214X NNK randomized library .............. 123
3-13 The results of screening the OYE 2.6 W244X NNK randomized library ........... 124
3-14 The results of screening the OYE 2.6 L260X NNK randomized library ............. 124
3-15 The results of screening the OYE 2.6 F307X NNK randomized library ............ 125
3-16 The results of screening the OYE 2.6 I311X NNK randomized library .............. 125
3-17 The results from the large scale screening assay............................................. 126
3-18 The results of the best mutants from all ten ISM-libraries ................................. 127
3-19 The results of the best mutants from OYE 2.6 E41X ........................................ 127
3-20 The results of the best mutants from OYE 2.6 D141X ...................................... 128
3-21 The results of the best mutants from OYE 2.6 E145X ...................................... 128
3-22 The results of the best mutants from all projects .............................................. 129
3-23 The positions targeted in both the ISM thermostability and local maximum projects ............................................................................................................. 130
3-24 The relative B-factor fractions from the OYE 2.6 D141E-S388P structure ....... 130
3-25 The relative B-factor fraction of all OYE 2.6 positions along the 3UPW structure ........................................................................................................... 131
3-26 The relative B-factor fraction of all OYE 2.6 positions along the 3TJL structure ........................................................................................................... 131
3-27 The B-factor fractions for all three published OYE 2.6 wt. structures ............... 132
3-28 The B-factor fractions of both the three OYE 2.6 wt. structures and the OYE 2.6 D141E-S388P structure .............................................................................. 132
3-29 The relative B-factor fractions of OYE 2.6 wt. in structure 3TJL ....................... 133
3-30 The relative B-factor fractions of OYE 2.6 D141E-S388P structure .................. 133
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3-31 The regeneration system used to make NADPH which reduces the FMN of OYE and allows the protein to turnover ............................................................ 134
4-1 Schematic illustration of the FMN environment in the active site of OYE homologs .......................................................................................................... 165
4-2 The mechanism of OYE 3 ................................................................................ 165
4-3 Diagram of the positions in the active site of OYE homologs ........................... 166
4-4 Loop 6 in OYE homologs .................................................................................. 167
4-5 List of OYE 3 substrates and reported conversion from the literature .............. 168
4-6 First set of substrates and theoretical binding mode products .......................... 177
4-7 Second set of substrates and theoretical binding mode products..................... 178
4-8 Third set of substrates and theoretical binding mode products......................... 179
4-9 The reactions used to test phenol binding by OYE 3 ........................................ 180
4-10 Crystals and crystallization conditions for of OYE 1, OYE 2.6, and OYE 3 ...... 180
4-11 The structure of OYE 3 ..................................................................................... 181
4-12 The active site for both OYE 1 and OYE 3 with bound FMN and substrate ..... 181
4-13 The active site for both OYE 3 and OYE 3 W116V with bound FMN and p-HBA .................................................................................................................. 182
4-14 The active site for both OYE 1 and OYE 1 F296S with bound FMN and p-HBA .................................................................................................................. 182
4-15 Results from screening the OYE 3 W116 site-saturation library against substrate 1 ........................................................................................................ 183
4-16 Results from screening the OYE 3 W116 site-saturation library against substrate 2 ........................................................................................................ 183
4-17 Results from screening the OYE 3 W116 site-saturation library against substrate 3 ........................................................................................................ 184
4-18 Results from screening the OYE 3 W116 site-saturation library against substrate 4 ........................................................................................................ 184
4-19 Results from screening the OYE 3 W116 site-saturation library against substrate 5 ........................................................................................................ 185
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4-20 Results from screening the OYE 3 W116 site-saturation library against substrate 11 ...................................................................................................... 185
4-21 Results from screening the OYE 3 W116 site-saturation library against substrate 13 ...................................................................................................... 186
4-22 Results from screening the OYE 3 W116 site-saturation library against substrate 14 ...................................................................................................... 186
4-23 Results from screening the OYE 3 W116 site-saturation library against substrate 15 ...................................................................................................... 187
4-24 Results from screening the OYE 3 W116 site-saturation library against substrate 21 ...................................................................................................... 187
4-25 Results from screening the OYE 3 W116 site-saturation library against substrate 22 ...................................................................................................... 188
4-26 Results from screening the OYE 3 W116 site-saturation library against substrate 23 ...................................................................................................... 188
4-27 Results from screening the OYE 3 W116 site-saturation library against substrate 24 ...................................................................................................... 189
4-28 Results from screening the OYE 3 W116 site-saturation library against substrate 25 ...................................................................................................... 189
5-1 The reaction of glycine and succinyl-CoA to make δ-AL using ALAS as a catalyst ............................................................................................................. 210
5-2 The proposed mechanism of ALAS .................................................................. 211
5-3 The active site of ALAS from R. capsulatus with glycine bound to PLP ........... 212
5-4 The active site of ALAS from R. capsulatus with succinyl-CoA ........................ 212
5-5 Reaction scheme of the derivatizing of δ-AL-pyrrole with Ehrlich’s reagent ..... 213
5-6 Reaction scheme of succinic anhydride with CoA to make succinyl-CoA ......... 213
5-7 The coupling of ALAS production of CoA from succinyl-CoA to α-Ketoglutarate Dehydrogenase production of NADH from NAD+ ....................... 214
5-8 Derivatization of amino acids using PITC ......................................................... 215
5-9 Derivatization of amino acids using MSTFA ..................................................... 215
5-10 The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent ................... 215
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5-11 The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent using a plate reader ...................................................................................................... 216
5-12 The reaction of succinyl-CoA with hydroxyl amine to displace the CoA. .......... 217
5-13 HPLC results from the reaction of succinic anhydride and CoA to make succinyl-CoA..................................................................................................... 217
5-14 HPLC results from the synthesis of succinyl-CoA from succinic anhydride and CoA co-eluted with 10x CoA ...................................................................... 218
5-15 HPLC results from the reaction of succinyl-CoA with hydroxylamine ............... 219
5-16 Results of the succinyl-CoA regeneration system ............................................ 220
5-17 Results of reactions from the succinyl-CoA regeneration system using mALAS and mALAS R433K ............................................................................. 221
5-18 Results of amino acid derivatization with MSTFA ............................................. 222
D-1 AZW2.Meth....................................................................................................... 246
D-2 AZW3.Meth....................................................................................................... 246
D-3 BTS2.Meth........................................................................................................ 247
D-4 BTS3.Meth........................................................................................................ 247
D-5 BTS4.Meth........................................................................................................ 248
D-6 BTS7.Meth........................................................................................................ 248
D-7 BTS8.Meth........................................................................................................ 249
D-8 FB1.Meth .......................................................................................................... 249
D-9 JON.Meth ......................................................................................................... 250
D-10 SEF.Meth ......................................................................................................... 250
D-11 YAP.Meth ......................................................................................................... 251
D-12 LMM.Meth......................................................................................................... 251
D-13 RWP2.Meth ...................................................................................................... 252
E-1 pET3b-OYE ...................................................................................................... 253
E-2 pBS2 ................................................................................................................. 253
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E-3 pFB1 ................................................................................................................. 254
E-4 pRP4 ................................................................................................................ 254
E-5 pGF23 .............................................................................................................. 255
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LIST OF ABBREVIATIONS
FPLC Fast Protein Liquid Chromatography
GC-FID Gas Chromatography Flame Ignition Detector
GC-MS Gas Chromatography Mass Spectrometry
HPLC High Pressure Liquid Chromatography
RMSD Root Mean Square Deviation
Y78X Y78 represents tyrosine at position 78 in the sequence of a protein. The X represents a random set of amino acids at that position that can include any, of the 20 amino acids.
Y78XKST Y78 represents tyrosine at position 78 in the sequence of a protein. The XKST represents a mutation at that position to a set of random amino acids which were obtained from a KST codon mix: alanine, cysteine, glycine, and serine.
Y78Xsm Y78 represents tyrosine at position 78 in the sequence of a protein. The Xsm represents a mutation at that position to a set of random amino acids which include the relatively small amino acids: alanine, cysteine, glycine, serine, threonine, and valine.
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
IMPROVING THE THERMOSTABILITY AND INCREASING THE SUBSTRATE RANGE OF OLD YELLOW ENZYME HOMOLOGS AND AMINOLEVULINIC ACID SYNTHASE
THROUGH PROTEIN ENGINEERING
By
Robert Wilson Powell III
December 2016
Chair: Jon Dale Stewart Major: Chemistry
We examined the consequences of mutating the general acid at position Y196 in
OYE 1. We screened a library of OYE 1 Y196 site-saturation mutants against substrates
of interest and discovered that only the cysteine variant gave an interesting result. We
then crystalized this mutant to determine how a cysteine was acting as a general acid.
We discovered that it was definitely not properly positioned, and the identity of the
actual general acid remains unknown.
We also examined an OYE homolog, OYE 2.6. Previous protein engineering
done by our group, as well as revelations from the crystal structure, gave us new insight
into ways to target the active site for mutagenesis. By making a matrix of small amino
acids mutantations at two neighboring positions, we hoped to open up the active site
and make it more amenable to alpha substituted 6 membered rings. We found that a
third mutation on the opposite side of the active site would give further improvement.
We then set out to improve the thermostability of OYE 2.6 by directed evolution.
We chose positions within OYE 2.6 that had high B-values in the crystal structure for
mutagenesis. After successfully increasing the thermostability of OYE 2.6, we then set
21
out to prove our hypothesis by crystallizing the thermostable mutant and comparing the
B-values at the mutated position. We found that we had indeed lowered the B-factor
value at the position which we mutated.
We next examined OYE 3 as a biocatalyst. We made a library of OYE 3 W116
mutants and screened it against several substrates of interest. We then set out to solve
the crystal structure of OYE 3 so that we could more appropriately examine the
structure of our mutants in complex with our substrates.
Lastly, we waded in mutagenesis of a PLP-dependent enzyme, mALAS. We
made three libraries of mALAS targeting positions T148, I151, and R85. We hoped that
these positions would be malleable positions in the active site that would allow us to
expand the substrate range of mALAS.
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CHAPTER 1 PROBING POSITION Y196 IN OLD YELLOW ENZYME 1
Background
Isolation and Purification of OYE
Old Yellow Enzyme (OYE) was first isolated by Warburg and Christian from
brewers’ bottom yeast (Saccharomyces carlsbergensis; subsequently re-named
Saccharomyces pastorianus) in 19321 while studying the oxidation of glucose-6-
phosphate (G-6-P) to 6-phospho-D-glucono-1,5-lactone. Glucose-6-phosphate
dehydrogenase (G6PDH) oxidized G-6-P using NADP+, which produced NADPH. OYE
was responsible for oxidizing NADPH by reducing O2 to H2O2 (Figure 1-1).2 The enzyme
was termed a ‘gelbe ferment’, or a yellow ferment. In 1938, Warburg and Has
discovered a second type of ‘gelbe ferment,’ leading them to designate the original
‘gelbe ferment’ as the ‘old yellow enzyme’ (OYE)3 and the enzyme has been known by
this name ever since. In 1955, Theorell and Åkeson purified the enzyme from the lysate
of brewers’ bottom yeast using a series of organic solvents and then crystallized the
protein using ammonium sulfate precipitation.4
Since its original purification in 1955, OYE has been the subject of significant
study, particularly by the late Professor Vincent Massey. In 1968, Massey and Matthews
discovered that after purification, oxidized OYE formed a charge transfer complex with
an unidentified “green forming compound”.5 The “green forming compound” lost affinity
for OYE when the FMN cofactor was reduced by sodium dithionite and removing this
“green forming compound” by dialysis further improved purification of the enzyme.
Massey and Abramovitz would later use phenol as a ‘green forming compound’ to bind
to the oxidized enzyme on a phenol column as the basis for a very efficient affinity
23
purification method for OYE.6 After binding, OYE could be eluted by in situ sodium
dithionite reduction. This affinity purification strategy significantly improved the isolation
of OYE and the same phenol columns are still widely used today for purification of OYE
and its homologs.
Several OYE isoforms exist in brewers’ bottom yeast and these complicated
efforts to obtain diffraction-quality crystals. In 1991, Massey and Saito solved this
problem by isolating the gene encoding OYE from Saccharomyces carlbergensis and
cloning it into an E. coli expression plasmid (pET3b).7 Subsequently, two additional
OYE-encoding genes were cloned from Saccharomyces cerevisiae, leading the initial
gene to be designated as OYE 1. Massey and Stott cloned and overexpressed OYE 2
in 19938 and OYE 3 was cloned and overexpressed in 1995 by Massey and Niino.9
Catalytic cycle and substrates of OYE
During their experiments to purify OYE through crystallization in 1955, Theorell
and Åkeson discovered that OYE had a flavin mononucleotide (FMN) cofactor.4 Later
studies by Massey et al. investigated the oxidative half reaction of OYE. These spectral
studies of the OYE charge transfer complex established the catalytic cycle of OYE and
have opened the way for it to be used as a biocatalyst ever since (Figure 1-2).8,10,11
While NADPH appears to be the physiological reductant for OYE, the native partner for
flavin re-oxidation has never been discovered.
Several studies have been performed with non-native flavin re-oxidation
substrates for OYE 1, some of the first involving quinones tested by Massey and Stott
during spectral studies.8 This suggested that OYE 1 could reduce electron-deficient
alkenes at the expense of NADPH, which touched off an avalanche of interest in using
this enzyme for asymmetric chemical synthesis. A vast array of alkene substrates have
24
been tested with OYE 1. These include many α,β-unsaturated ketones, aldehydes,10–14
nitroalkenes,15–18 and even alkynes (Figure 1-3).19
Structure and Mechanism of OYE
Though OYE 1 was first purified and crystallized from lysate by Theorell and
Åkeson in 1955, its three-dimensional structure was not determined until 1994 when
Karplus and Fox successfully obtained diffraction quality crystals of OYE 1.4,20 The
overall structure of OYE 1 features an α/β-barrel (TIM barrel) with the active site buried
in the barrel along with the FMN cofactor. Within the active site, T37, G72, Q114, and
R243 form hydrogen bonds with the FMN and lock it into place (Figure 1-4). In 1998,
Massey and Brown used site directed mutagenesis and a series kinetic experiments to
establish the role of H191 and N194.21 These amino acids stabilize the oxyanion
intermediate prior to its protonation. In the same year, Massey and Kohli also examined
the role of Y196. Site directed mutagenesis was used to prepare a phenylalanine
substitution and the kinetic properties of both it as well as the wild type protein were
compared. Interestingly, the Y196F mutation had little effect on ligand binding; however,
the oxidative half reduction was nearly 6 orders of magnitude slower than the wild-
type.11 This study helped establish tyrosine as the general acid in the catalytic
mechanism of OYE 1 (Figure 1-5).
Our group has investigated the potential of OYE 1 as a biocatalyst.13,22,23 Much of
our work has been focused on preparing and testing various OYE 1 mutants for their
potential to accept synthetically interesting substrates. Amino acids mentioned above
are essential for FMN or substrate binding and / or catalysis and for this reason, were
not subjected to mutagenesis (Figure 1-7). On the other hand, we found that W116
25
accepted a wide variety of substitutions, some of which allowed for alternative (“flipped”)
substrate binding modes that led to opposite stereopreference (Figure 1-6).22–25
Project Overview
While Y196 was shown to be critical for the oxidation half-reaction and
associated with oxyanion protonation (Figure 1-5), we hypothesized that other residues
with even greater acidity might yield more efficient OYE 1 variants. We therefore
explored site-saturation mutagenesis of Y196 in OYE 1. A randomized library variants at
this position was created and screened against two alkene substrates (11 and 12 shown
in Figure 1-10). After finding catalytically active members in the library, plasmids were
sequenced and the final variant not already present was added. The complete Y196
site-saturation mutagenesis library was then screened against a broader panel of
substrates (Figures 1-8 through 1-10). These efforts revealed that the Y196C mutant
was catalytically active against a subset of the substrate collection. This was
reminiscent of the Y193C variant of Pichia stipitis OYE 2.6, a fascinating, if inconsistent,
mutant discovered in a parallel project by Adam Walton. To determine how a cysteine at
position 196 of OYE 1 could lead to substrate protonation, we determined the crystal
structure of this variant. We hoped that the information from this structure might shed
light on the analogous Y193C mutant of P. stipitis OYE 2.6 that has stubbornly resisted
all efforts at crystallization.
Results and Discussion
OYE 1 YI96 Site-Saturation Mutagenesis Library
We chose to prepare the site-saturation library en masse, deferring DNA
sequencing until it became clear that one or more active variants were actually present.
To this end, a randomized library of OYE 1 Y196 was made using a pair of primers with
26
a NNK mix at the codon for Y196 (N = any base, K = G / T). This base doping scheme
encompasses 32 different codons that includes all 20 amino acids plus one stop codon.
Using a variation of a cloning method reported by Zheng et al. that our group developed
for cloning NNK randomized libraries,26,27 PCR was performed and a pooled plasmid
sample obtained from transformants was sequenced. Using a method for evaluating
libraries developed in our lab by Sullivan and Walton,26 the plasmid mix was evaluated
for codon degeneracy. Sequencing of this pooled sample revealed a plasmid mix a
codon mix that gave a Q score of 0.85 (Figure 1-11), which implied that all 19 mutants
could be obtained with subsequent transformation into an E. coli overexpression strain.
A randomized library of 95 individual OYE 1 Y196 mutants (plus the wild-type control)
was assembled from transformants derived from the pooled plasmid sample. Each
variant was screened against 2-methyl-2-cyclohexen-1-one (substrate 12) to detect
catalytic activity. Plasmid DNAs from hits in this initial screen were sequenced to
determine the codon at position 196. The best variant was Y196C. This was fascinating
since an analogous mutant from P. stipitis OYE 2.6 (Y193C) also proved to be the
optimal substitution for the catalytic Tyr side-chain.28
These initial results were sufficiently promising to justify sequencing the
randomized library to determine whether all of the position 196 variants were actually
present and identify any that needed to be added to complete the set. These efforts
revealed 18 / 19 mutants, with only OYE 1 Y196W being absent. The original Q score
predicted that all 19 variants were present in the plasmid mix used to transform the E.
coli overexpression strain. Our obtaining 18 mutants was therefore very encouraging,
further underscoring the value and accuracy of our simple library quality control
27
determination. The missing OYE 1 Y196W mutant was made by standard methods,
then and the complete library was then used to screen other substrates (Figures 1-12
through 1-18). Unfortunately, none of the Y196 variants improved conversion for either
the Baylis-Hillman adducts (1 – 3) or either carvone enantiomer (7 and 8), with no
conversion for the former and minimal conversion for the latter. Also, no mutant gave
any activity against substrate 11, a screening substrate containing a 5-membered ring.
As expected from our initial screening efforts, we observed minor conversion for
substrate 12. The best variant (Y196C) gave racemic reduction product from 12 (wild-
type gives >98% ee (R)), although the conversion was only 20%.
Crystal Structure of OYE 1 YI96C
Given the difference in spatial locations between the acidic protons of a Tyr and
Cys residue, it was not clear how the Y196C mutant retained catalytic activity. We
therefore crystallized this OYE 1 variant in the hopes that the structure would provide
insight. 4-Hydroxybenzaldehyde (p-HBA) is an OYE inhibitor that binds strongly and can
provide information on the active site environment. We therefore soaked the mutant
crystals with p-HBA.
The OYE 1 Y196C mutant structure was near identical to the OYE 1 wt. structure
(1OYB) with a RMSD of 0.29 Å. The active site aligned nearly completely as well, with
the only difference being the Y196C mutation. Surprisingly, the structure revealed that
cysteine was not properly placed to participate directly in the alkene reduction step, as
its thiol moiety was directed away from the active site (Figure 1-19). In fact, the β-
carbon of the cysteine was 0.6 Å further than the tyrosine to the C2 of the p-HBA and
the sulfur was 5.0 Å further than the oxygen of the tyrosine. The thiol did not form a
disulfide bridge with a neighboring residue nor did it have an alternate oxidation state
28
like sulfenic (R-SOH), sulfinic (R-SO2H), or sulfonic acid (R-SO3H). This implies that a
general acid other than the Cys side-chain at position 196 acts as the general acid. The
actual proton source remains unknown, although it is intriguing that alkene 12 was
reduced to racemic product, consistent with solvent protonation after dissociation of the
enol(ate). On the other hand, this does not explain why the structurally analogous
carvone enantiomers were not accepted by the Y196C variant. While OYE is able to
tolerate the cysteine substitution, this change seriously impairs its function.
Experimental
General
Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained
from Integrated DNA Technologies (IDT). All other reagents were obtained from
commercial suppliers and used as received. Plasmids were purified on small scales by
Wizard® minicolumns (Promega Life Sciences) and on large scales using CsCl density
gradient ultracentrifugation.29 DNA sequencing was carried out by the University of
Florida ICBR using capillary fluorescence methods using standard protocols. LB
medium contained 5 g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.
ZY medium contained 5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052
contained 25% glycerol, 2.5% glucose, and 10% a-lactose monohydrate. NPS x20
contained 66 g/L (NH4)2SO4, 136 g/L KH2PO4, and 142 g/L Na2HPO4.
29
Cloning
Construction of plasmids used to make the OYE 1 Y196 library
The plasmid used as a template to make the OYE 1 Y196 library was pET3b-
OYE (Appendix E Figure E-1), a pET3b derivative containing the gene for OYE 1.7
pET3b-OYE was originally a gift from Dr. Betty Jo Brown (University of Michigan).
Construction of mutants in the OYE 1 Y196X randomized library
All PCR samples were purified using Wizard® Plus SV Gel PCR Clean up kits by
Promega according to the manufacturer’s instructions. Samples were then incubated
overnight with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent
template. The first portion of DpnI was added immediately after PCR clean up and the
second was added after 4 hours of digestion. After DpnI digestion, samples were
purified using Wizard® Plus SV Gel PCR Clean up kits by Promega.
Digested PCR samples were used to transform ElectroTen-Blue®
electrocompetent cells (ETB) using a Gene Pulser® from BioRad. Electroporation was
carried out with 4 μL of PCR sample and 50 μL of ETB cells using 2.5 kV.
Electroporated samples were incubated with 600 μL of SOC media at 37°C for 1 h.
Cells were then plated onto LB-amp agar plates and grown at 37°C for 36 h. The best
results were obtained from ETB daughter cells grown from the commercial stock on the
same day as the transformation would take place. Granddaughter cells provided fewer
transformants. Transformed cells were then pooled by rinsing the plate with a minimal
volume of LB and scraping with a rubber policeman. Plasmid DNA was extracted and
purified using Wizard® Plus minipreps DNA purification system by Promega according
to the manufacturer’s instructions. Purified, pooled plasmids were sequenced by ICBR
using Sanger sequencing. Raw electropherograms (chromat files) obtained from Sanger
30
sequencing were analyzed to estimate the samples degeneracy. Degeneracy could be
gauged for samples using a NNK primer mix (Figure 1-11) by the method developed by
Sullivan and Walton.26 This gave a Q-score of 0.85, suggesting that all position 196
variants were present in the library. A 4 μL aliquot of the purified, pooled plasmid was
used to transform overexpression strain E. coli BL21 (DE3) Gold cells using
electroporation (2.5 kV). Three transformants per possible codon would be required to
obtain a good representation for each possible codon in a pooled plasmid sample. For
this reason, 95 randomly-chosen colonies were used to seed 600 μL of LB-amp in a 96
well plate. Well H12 was reserved for the wild-type control. The plate was shaken
overnight at 37ºC to reach saturation. The library was stored by mixing 120 μL of each
culture with 30 μL of sterile 80% glycerol in a fresh 96 well plate. This gave a final
glycerol concentration of 15%, allowing the plate to be stored indefinitely at -80ºC.
Substrates
A list of substrates and products is shown if Figure 1-8 through 1-10.
2-(Hydroxymethyl)-cyclopent-2-enone (1)
2-(Hydroxymethyl)-cyclopent-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Kar and Argade.30 2-(Hydroxymethyl)-
cyclopent-2-enone can be detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm × 30 m). The temperature program used began with an initial
temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature
of 180°C at which the program remained for 5 min (GC method is listed as AZW2.Meth
in Appendix D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The
reduced products (S)- and (R)-6 eluted near 11.4 and 10.2 min, respectively.
31
2-(Hydroxymethyl)-cyclohex-2-enone (2)
2-(Hydroxymethyl)-cyclohex-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Rezgui and El Gaied.31 2-(Hydroxymethyl)-
cyclohex-2-enone was detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm × 30 m). The temperature program used began with an initial
temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature
of 180°C at which the program remained for 5 min (GC method is listed as AZW2.Meth
in Appendix D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The
reduced products (S)- and (R)-7 eluted near 10.2 and 10.8 min, respectively.
Methyl 2-(hydroxymethyl)acrylate (3)
Methyl 2-(hydroxymethyl)acrylate was prepared in our lab by Bradford Sullivan28
using the method developed by Turki et al.32 Methyl 2-(hydroxymethyl)acrylate was
detected during screening by GC-FID using a Beta Dex 225 column (0.25 mm × 30 m).
The temperature program began with an initial temperature of 100°C for 12 min,
followed by an increase at 20°C/min to a temperature of 180°C at which the program
remained for 5 min (GC method is listed as AZW3.Met in Appendix D). Methyl 2-
(hydroxymethyl)acrylate eluted near 11.8 min. The reduced products (S)- and (R)-7
eluted near 10.7 and 11.3 min, respectively.
(S)-(+)-Carvone (7)
(S)-(+)-Carvone was purchased from Sigma Aldrich and it can be detected by
GC-MS using a DB-17 column (0.25 mm × 30 m). The temperature program used
began with an initial temperature of 60°C for 2 min, followed by an increase at 10°C/min
to a temperature of 195°C at which the program remained for 10 min (GC method is
listed as JON.Meth in Appendix D). (S)-(+)-Carvone eluted near 12.5 min. A mixture of
32
reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a standard to assign
the peaks for both cis- and trans-9 (11.7 and 11.3 min, respectively).
(R)-(-)-Carvone (8)
(R)-(-)-Carvone was purchased from Sigma Aldrich and it can be detected during
screening by GC-MS using a DB-17 column (0.25 mm × 30 m). The temperature
program began with an initial temperature of 60°C for 2 min, followed by an increase at
10°C/min to a temperature of 195°C at which the program remained for 10 min (GC
method is listed as JON.Meth in Appendix D). (R)-(-)-Carvone eluted near 12.5 min. A
mixture reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a standard
to assign the peaks for both cis- and trans-10 (11.7. and 11.3 min, respectively).
2-Methyl-2-cyclopenten-1-one (11)
2-Methyl-2-cyclopenten-1-one was purchased from Sigma Aldrich. 2-methyl-2-
cyclopenten-1-one was detected during screening by GC-MS using a DB-17 column
(0.25 mm × 30 m). The temperature program began with an initial temperature of 60°C
for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the
program remained for 10 min (GC method is listed as JON.Meth in Appendix D). 2-
methyl-cyclopenten-1-one eluted near 7.0 min, and the reduced product 13 eluted near
5.2 min.
2-Methyl-2-cyclohexen-1-one (12)
2-Methyl-2-cyclohexen-1-one was purchased from Sigma Aldrich. 2-methyl-2-
cyclohexen-1-one was detected during screening by GC-MS using a DB-17 column
(0.25 mm × 30 m). The temperature program began with an initial temperature of 60°C
for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the
program remained for 10 min (GC method is listed as JON.Meth in Appendix D). 2-
33
methyl-2-cyclohexen-1-one eluted near 8.5 min and the reduced product 14 eluted near
7.2 min.
Screening Assay
E. coli BL21 (DE3) Gold cells harboring plasmids containing OYE 1 Y196
mutants of interest were grown in a 96 well plate containing 600 μL of LB-amp. Cells
were grown at 37°C with 250 rpm of agitation overnight. The saturated cultures were
then used to inoculate a larger 2 mL square bottom 96 well plate. This larger square
bottom plate contained 600 μL of an auto-induction media. The auto-induction medium
contained a mix of ZY media, 50x5052, 20x NPS, and 200 μg/mL ampicillin.33 Cells
were induced in an aeration case developed at the University of Florida by the machine
shop in the Department of Chemistry. Induction occurred at 37°C with 350 rpm of
agitation overnight. The increased agitation is required for induction to occur. Induced
cells were then separated from the auto-induction medium by centrifugation. The
supernatant was removed and the induced, pelleted cells were resuspended in 600 μL
of reaction mixture, which contained 50 mM KPi, 100 mM glucose and 15 mM alkene
substrate, pH 7.0. Reactions were shaken at 250 rpm overnight at room temperature
before quenching by adding 500 μL of ethyl acetate. The organic phase was separated
by centrifugation and analyzed by GC.
Protein Purification and Crystallogenesis of Y196C OYE 1
The Y196C OYE 1 mutant was purified by the same methods used previously for
wild-type and mutant OYE 1,34 which is a modification of the procedure originally
developed by Massey.6 E. coli BL21 (DE3) Gold cells harboring pET3b-OYE 1 Y196C
were grown at 37°C in a 4 L New Brunswick Scientific M19 fermenter containing LB-
amp. Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid
34
log phase. Protein overproduction was induced by adding IPTG and glucose to final
concentrations of 0.4 mM and 100 mM, respectively. The culture was grown at 30°C
with 600 rpm of agitation for 4 h. The culture was then chilled at 4°C for 30 min before
centrifugation at 5,000 × g. The wet cell pellet was then resuspended in 100 mM Tris-Cl
buffer containing 10 μM phenylmethane sulfonyl fluoride (PMSF) at pH 8.0. Cells were
then lysed under 12,000 psi with the aid of a French press. Cell extract was centrifuged
at 18,000 × g for 1 h to remove insoluble debris. Nucleotides were precipitated by
adding protamine sulfate to a final concentration of 1 mg/mL and stirring at 4°C for 20
min. The supernatant was separated by centrifugation at 18,000 × g for 20 min. Protein
was precipitated out of solution by adding solid ammonium sulfate in 5 portions every 5
min to achieve a final concentration of 78% saturation. The protein precipitate was then
separated by centrifugation at 18,000 × g for 1 h.
Purification of OYE 1 by an N-(4-hydroxybenzoyl) aminohexyl agarose affinity
column requires that the active site be emptied of any bound ligand that would interfere
with binding to the phenol moiety of the column matrix. This was accomplished by
successive buffer exchanges during dialysis. The ammonium sulfate pellet obtained
from the salt cut was resuspended in 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM
PMSF, pH 8.0. This was dialyzed against 1 L of 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10
μM PMSF, pH 8.0 overnight at 4ºC. The sample was then dialyzed against 1 L of the
same buffer containing 10 mM sodium dithionite for 2 h at 4ºC. After 2 h, the buffer was
exchanged for a fresh 1 L of buffer containing 10 mM sodium dithionite and dialysis
continued for 2 h. The sample was then transferred to a fresh 1 L of buffer without
dithionite and dialyzed for 2 h, after which, the buffer was exchanged with a final 1 L of
35
fresh buffer and dialyzed overnight. The final sample was then centrifuged at 18,000 × g
for 30 min to remove any insoluble debris accumulated during dialysis.
Dialyzed protein samples were loaded onto the affinity column in 10 mL portions.
The affinity column was equilibrated with 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM
PMSF, pH 8.0. Binding of OYE 1 turned the column green. After washing with 30 mL of
starting buffer, the desired protein was eluted by washing with 100 mM Tris-Cl, 100 mM
(NH4)2SO4, 10 μM PMSF, 4 mM sodium dithionite, pH 8.0. OYE 1 Y196C was then
further purified by gel filtration with a Superdex 200 column (Pharmacia) using 50 mM
Tris-Cl, 50 mM NaCl, pH 7.5. Pooled fractions containing the desired protein were then
concentrated by ultrafiltration using an Amicon centrifugation tube to a final
concentration of 20 mg/mL. Protein concentration was determined by absorbance at
280 nm using an extinction coefficient (ε) and molecular weight (MW) estimated by
protparam (OYE 1 Y196C had an ε of 70,820 M-1cm-1 with a MW of 44,954 Da).35
Crystals were grown using the published conditions discovered by Fox and
Karplus.20 Wells contained 6 μL of 20 mg/mL protein in 50 mM Tris-Cl, 50 mM NaCl, pH
7.5 and used hanging drop vapor diffusion. The crystallization solution contained 35%
(v/v) PEG 400, 100 mM Na HEPES, 200 mM MgCl2, pH 8.3. The best crystals obtained
were obtained after 10 days at 6°C. After crystallization, crystals were mounted in
appropriate loops and soaked with p-HBA before being flash cooled in liquid nitrogen
and sent for data collection. No cryoprotectant was used.
The Data Collection and Crystal Structure of OYE 1 Y196 Mutants
The best crystals diffracted to a maximum usable resolution of 1.36Å using the
X6A beamline at Brookhaven National Laboratory. The unit cell measured was 141.1
141.1 42.8 Å 90 90 90 and the crystals belonged to space group P 43 21 2. The
36
asymmetric unit contained 1 molecule with a solvent content of 48.05% and a Matthew’s
coefficient of 2.37 Å3/Dal.36
Reflection data were processed using the iMOSFLM program from the CCP4
program suite to a resolution of 1.36Å.37 Phases were obtained using the Phaser-MR
utility of the PHENIX program suite by molecular replacement using a modification of S.
pastorianus OYE 1 (PDB code 1OYB) as the search model.38 All ligands and water
molecules were removed prior to molecular replacement. Inspection of the model
showed one OYE 1 chain present in the asymmetric unit. The best solution for the
space group was determined to be P 43 21 2. The initially calculated 2Fo-Fc and Fo-Fc
maps showed electron density patterns that could be easily identified as the FMN
cofactor. The FMN cofactor was modeled into the structure. Initial refinement using the
xyz coordinates, B-factors, real-space, and occupancies refinement strategy features in
PHENIX refine as well as continued cycles of model building with the aid of the structure
validation tools in COOT produced a model with an Rfree of 0.1824.39 Continual
iterations of using the structural validation tools in COOT and PHENIX.refine produced
a model with an Rfree of 0.1698. At this point the p-HBA was modeled into the active site
and subsequent rounds of model building with COOT and refinement produced an Rfree
of 0.1554.
Conclusions
The Y196 mutants of OYE 1 showed no improvement of product range for the
enzyme. However, the observation that Y196 could be successfully replaced by Cys is
intriguing since the crystal structure of the mutant with bound p-HBA showed that the
cysteine residue was not appropriately positioned to act as a general acid. Our current
37
hypothesis is that solvent supplies the proton to the enol(ate) formed by enone
reduction, possibly after dissociation from the active site.
These OYE 1 structural data may also provide guidance for the analogous
Y193C mutant of P. stipitis OYE 2.6. The latter could not be crystallized because the
protein could not be purified with reproducible properties. Like OYE 1, Cys was the sole
functional replacement for Tyr in P. stipitis OYE 2.6. Whether the structure and catalytic
mechanism of this mutant resembles its OYE 1 counterpart remains to be determined.
38
Figure 1-1. The catalytic cycle of G6PDH investigated by Warburg and Christian.1,28
Figure 1-2. The catalytic cycle of OYE 1 established by Massey and Vas.10,40
39
Stott et al. (1993)8
Vaz et al. (1995)10
Figure 1-3. List of OYE 1 substrates from the literature.
40
Vaz et al. (1995)10
Figure 1-3. (Continued).
41
Vaz et al. (1995)10
Kohli et al. (1998)11
Meah et al. (2000)15
Figure 1-3. (Continued).
42
Meah et al. (2001)16
Williams et al. (2004)17
Swiderska et al. (October 2006)12
Figure 1-3. (Continued).
43
Swiderska et al. (December 2006)18
Mueller et al. (March 2007) 19 Bougioukou et al. (2008)13
Mueller et al. (September 2007)41
Figure 1-3. (Continued).
44
Hall et al. (2008)42
Padhi et al. (2009)23
Winkler et al. (2010)43
Figure 1-3. (Continued).
45
Stueckler et al. (September 2010)14
Stueckler et al. (October 2010)44
Brenna et al. (June 2011)45
Figure 1-3. (Continued).
46
Brenna et al. (July 2011)46
Brenna et al. (December 2011)47
Brenna et al. (2012)48
Durchschein et al. (2012)49
Figure 1-3. (Continued).
47
Tasnadi et al. (March 2012)50
Tasnadi et al. (June 2012)51
Figure 1-3. (Continued).
48
Brenna et al. (2013)52
Brenna et al. (January 2014)53
Figure 1-3. (Continued).
49
Brenna et al. (July 2014)54
Turrini et al. (2015)55
Figure 1-3. (Continued).
50
Figure 1-4. FMN Diagram displaying the FMN (gold) environment in the active site of OYE 1. OYE 1 (green) uses hydrogen bonding partners to lock FMN in place within the active site (T37, G72, Q114, and R243). OYE 1 also uses the hydrophobic bonding partners beneath to FMN shown with blue circles (P35, L36, and I351) to lock it into position.
Figure 1-5. Catalytic mechanism of OYE 1. Mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 1 (green). OYE 1 uses positions H191 and N194 as hydrogen bonding partners to lock the carbonyl into position.
51
Figure 1-6. OYE substrate binding modes. In both binding modes, cyclohexenone docks in the active site above and parallel to the plane of the reduced FMN. The carbonyl oxygen forms hydrogen bonds with the residues N194 and H191. The hydride from N5 is transferred to the electron deficient β-carbon. Y196 acts as a general acid to protonate the resulting enolate (see figure 1-5) at the α-carbon. The “flipped” conformation requires a shift in the angle of hydride and proton transfer and is sterically crowded by the presence of Trp116. The stereochemistry of each binding mode product is indicated with the protic hydrogen (green) and the hydride hydrogen (gold) below each scheme. For a prochiral substrate the two binding modes determine product stereochemistry.28
52
Figure 1-7. OYE 1 active site diagram. This diagram shows the areas of the OYE 1
active site. The orange section contains the “east side” of the active site which will be discussed more in chapter 2. The magenta section contains other active site positions which form a gate with the positions on loop 6. Loop 6 is the mobile part of this gate and opens up to allow NADPH to enter the active site and reduce oxidized FMN. The green section includes position on loop 6 which will be of discussed more in chapter 4. The Blue segment contains the “west side” of the active site which will be discussed more in Chapter 2. The yellow segment contains positions that interact with the bound substrate (N194 and H191) and position Y196 which acts as a general acid in OYE 1 catalysis.
53
Figure 1-8. List of Baylis-Hillman substrates screened by OYE 1 Y196 library.
54
Figure 1-9. List of carvone substrates screened by OYE 1 Y196 library.
55
Figure 1-10. List of screening substrates screened by OYE 1 Y196 library.
56
Figure 1-11. Calculations for obtaining a Qscore of a pooled plasmid mix from a NNK primer mix using data from a theoretical sequencing electropherogram. This figure shows a theoretical electropherogram with theoretical peak heights. a) The fraction of each peak height at a position over all peak heights at that position is shown on the left side of each peak. Peaks correspond to: blue/thymine, orange/guanine, green/adenine, purple/cytosine, and the dashed black represents perfect degeneracy. b) The peak fraction of each base is used to estimate the amount of codons containing that base at that position. c) The sum of those estimates are used to obtain a Q value (QN or QK) for that position. d) The sum of the weighted QN and QK values is used to calculate the Qscore for the pooled mix. The Qscore approaches perfect degeneracy (equal amounts of bases at each position) as it approaches 1.0.26
57
Figure 1-12. OYE 1 Y196 library screening results for 1. No conversion was observed for any mutants. OYE wt. results are consistent with previous findings.22
Figure 1-13. OYE 1 Y196 library screening results for 2. No conversion was observed for any mutants. OYE wt. results are consistent with previous findings.22
58
Figure 1-14. OYE 1 Y196 library screening results for 3. No conversion was observed for any mutants. OYE wt. results are consistent with previous findings.22
Figure 1-15. OYE 1 Y196 library screening results for 7. OYE wt. results for (S)-(+)-carvone are consistent with previous findings.24
59
Figure 1-16. OYE 1 Y196 library screening results for 8. OYE wt. results are consistent with previous findings.24
Figure 1-17. OYE 1 Y196 library screening results for 11. The stereochemistry of products for substrate 11 was not evaluated during screening.
60
Figure 1-18. OYE 1 Y196 library screening results for 12. The stereochemistry of products for substrate 12 was not evaluated during screening. The stereochemistry of OYE 1 Y196C was evaluated after initial screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m) using AZW2.Meth (Appendix D). Peaks were never assigned.
Figure 1-19. Alignment of OYE 1 wt. and OYE 1 Y196C. This figure shows OYE 1 wt. (green) aligned to and OYE 1 Y196C (orange) (PDB ID 1OYB) with FMN cofactor (yellow). Both structures have p-HBA bound in the active site.20
61
CHAPTER 2
IMPROVING THE PRODUCT RANGE OF OYE 1 AND OYE 2.6 THROUGH PROTEIN ENGINEERING
Background
Mutagenesis of the East Side of the Active Site in OYE
Given the potential of alkene reductases for making products with high
enantiomeric excess, we were eager to explore this class of enzymes for use as a
biocatalysts. Long before we waded into an expansive mutagenesis projects which are
frequent in this group now, we wanted to probe a set of alkene reductases against a
series of substrates. To this end, our lab made an alkene reductase library with 16
alkene reductases.56 This collection of alkene reductases was assembled by Despina
Bougioukou and was screened against a series of substrates of interest.56 Table 2-1
lists all the fully sequenced alkene reductase libraries made by the Stewart group.
One enzyme in that library that was of interest to our group was Old Yellow
Enzyme (OYE 1) from Saccharomyces pastorianus. Using the crystal structure of OYE
1 solved by Fox and Karplus,20 our group identified a position in the active site that we
believed might affect the orientation of substrate binding and could influence the type of
products we could obtain. Position W116 is located on the east side of the OYE 1 active
site and the tryptophan at this position is in close proximity to groups that extend off the
alpha carbon of any bound carbonyl substrate. We believed that substitution of this
bulky tryptophan for a different residue may allow substrates to bind in an alternate
“flipped binding mode”. Figure 2-1 shows the flipped binding mode in the OYE 1 active
site. Binding in this mode would give the alternate enantiomer at the alpha carbon
following catalysis. To pursue this idea, in 2009 Despina Bougioukou and Santosh
62
Padhi used site saturation mutagenesis at position W116 of OYE 1 to discover new
mutants that would allow flipped binding.23 Our group was very interested in a set of
Baylis-Hillman substrates (substrates 1-3) as well as a pair of carvone substrates
(substrates 7 & 8) (Figure 2-2). Bougioukou and Padhi screened the carvone substrates
against the OYE 1 W116 mutants. The blind screening done with these mutants
identified functional mutations including OYE 1 W116F, W116I, W116L, W116M and
W116Y. It was also discovered that OYE 1 W116I would flip (S)-(+)-carvone and give
the trans product, trans-9 (diasteriomeric excess (de) = 88% in favor of trans and
conversion was 98%). OYE 1 wild type (wt) however, gave the cis product (cis-9) for
(S)-(+)-carvone (de = 93% in favor of cis and conversion was 48%). These findings
were significant enough to warrant making a full degenerate library containing all 20
residues substituted at position W116. In 2011, Adam Walton and coworkers screened
the Baylis-Hillman substrates against this OYE 1 W116 library.22 They found that some
of these mutants would allow flipped binding for 2-(hydroxymethyl)-cyclopent-2-enone
(substrate 1) and methyl 2-(hydroxymethyl)acrylate (substrate 3). In 2013, Yuri Pompeu
and Bradford Sullivan screened the carvone substrates against the completed OYE 1
W116 mutants.24 They identified new mutants that could provide the alternate trans
product (trans-9) for (S)-(+)-carvone (OYE 1 W116A, W116C, W116E, W116G, W116I,
W116M, W116N, W116Q, W116S, W116T, and W116V) as well as the alternate cis
product (cis-10) for (R)-(-)-carvone OYE 1 W116A, and W116V). Furthermore, the
crystallographic studies done by Pompeu and Sullivan showed (S)-(+)-carvone bound in
the flipped binding mode within the active site of the OYE 1 W116I mutant. Figure 2-3
shows the active site of OYE 1 W116I with (S)-(+)-carvone bound.24 And as such, this
63
work provided firm evidence for the idea that position W116 played a role in
discriminating substrates during binding and that mutating this position would make the
active site more amenable to a flipped binding mode which could alter the
stereochemistry of the products.
Another OYE homolog that our group investigated was OYE 2.6 from Pichia
stipitis. OYE 2.6 has an isoleucine at position I113 which is the analogous position to
W116 in OYE 1. What is fascinating about OYE 2.6 is that it gives the same cis product
(cis-9) as OYE 1 wt for (S)-(+)-carvone. This is noteworthy because when these two
homologs have the same residue at an analogous position they give different products.
Because of this, our group began focusing on OYE 2.6 as a biocataylst.34,57,58 In one of
our most ambitious mutagenesis projects to date, our group extensively mutated several
positions in the OYE 2.6 active site to explore its potential for engineered
biocatalysis.34,57,58 The approach our group used during this project was a technique
developed by Manfred Reetz called iterative saturation mutagenesis (ISM).59 ISM is a
progressive protein engineering strategy that targets several positions of interest over a
series of mutagenic rounds. The best mutant identified for each position is then used as
an “anchor” for a second round of mutagenesis where a different position is
randomized. Successive rounds are carried out with the best mutant at each position
being added to the next round of randomization. The goal of this strategy is to hone in
on multi-mutated variants with exceptional properties. Targeting positions around the
OYE 2.6 active site, our lab made several 1st, 2nd, and 3rd generation libraries.57 Figure
2-4 has a diagram showing all the residues in the OYE 2.6 active site. The ISM
experiments were concluded after variants were discovered that would allow the flipped
64
binding mode and give the (R)- product with high enantiomeric excess (ee) for two of
the three Baylis-Hillman substrates. These variants worked for both substrate 3 and
substrate 1.57 A 2nd generation library uncovered an OYE 2.6 Y78W / F247A double
mutant that gave the best results for the 2-(hydroxymethyl)-cyclohex-2-enone (substrate
2). Table 2-2 summarizes the best variants of OYE 1 and OYE 2.6 for obtaining de and
ee for carvone and Baylis-Hillman substrates. The best result however was a
conversion 43% and an ee of 37% (S), which is not ideal since the product of interest is
the R enantiomer ((R)-5). As such, a good solution for obtaining the (R)-5 product from
2 was not discovered during the ISM project.
Project Summary
Though an enzyme that would exclusively make the (R)-5 product from 2 was
never discovered, we did find a set of promising positions that gave us a good idea on
how to move forward. Since OYE 1 wt only gives 10% conversion for 2 we decided to
focus our efforts on engineering OYE 2.6 which gives nearly 100% conversion. We
believed that the answer would be found at the east side of the active site. Molecular
modeling shows that the alternate flipped binding mode of 2 would crash into the east
side residues in OYE 2.6. Figure 2-7 shows OYE 2.6 with a 6-membered ring modeled
into the active site. The best double mutant combination identified during the ISM
project with a pair of east side mutants, was OYE 2.6 Y78W / I113C. This double
mutant includes two positions that are located on the east side of the active site and
their residues extend into the space where any substrate that attempts a flipped binding
to OYE 2.6 would occupy. Figure 2-6 shows the crystal structure of OYE 2.6 Y78W /
I113C with malonate bound into active site. The best triple mutant discovered for
obtaining the (R)-5 product from 2 was OYE 2.6 Y78W / I113C / F247A which includes
65
the two mutants at positions on the east side of the OYE 2.6 active site of interest as
well as one position on the west side.57 Given the results of these variants, we wanted
to further investigate these three positions.
The main aim of this project was to find a combination of mutations that would
improve the yield for the (R)-5 product for OYE 2.6 and in doing so, hopefully discover
new biocatalysts that could be used on other future substrates. In this project we
examine what would happen if we compacted the large obstructive steric bulk on the
east side of the active site in OYE 2.6. We began by making a randomized matrix library
of the two positions on the east side of the active site that had the most notable effect
on enantiomeric excess during the ISM experiments, positions Y78 and I113. These
positions would be replaced with a pair of smaller residues; alanine, cysteine, glycine,
serine, threonine, and valine. We then assembled a library with presequenced double
mutants at those positions. Since the best double and triple mutant variants contained
mutations at position F247, that was another position we wanted to target during this
project. Since we believe that position Y78 and I113 interact with each other, it was
important to find the right combinations at these two positions before looking at the
F247 position. After anchoring off the best double mutants discovered during the
mutagenesis of the east side of active site, a set of 2nd round libraries were made
targeting the F247 position located on the west side of the active site.
Results and Discussion
OYE 2.6 Y78Xsm / I113Xsm Randomized Library
Initial cloning simultaneously targeted two positions located on the east side of
the OYE 2.6 active site, a tyrosine at position Y78 and an isoleucine at positon I113.
The aim of this strategy was to replace these two residues with a pair of small amino
66
acids which would take up less space and allow the alternate flipped binding mode for 2
which would in turn produce more (R)-5 product. The smaller pair of residues would
include a combination of either alanine, cysteine, glycine, serine, threonine or valine.
Given the substantial amount of cloning that had already been done on this enzyme
(fourteen 1st generation, nine 2nd generation, and two 3rd generation libraries)28,57 it was
preferable to survey the active site with as minimal effort as possible. To minimize the
laborious task of cloning a matrix of double mutants, a randomized cloning approach
was chosen to assemble the first library. A KST random primer mix contains an equal
number of codons for alanine, cysteine, glycine, and serine. Anchoring off a pBS2
vector, a pET derivative with GST-OYE 2.6 fusion protein (Appendix E, Figure E-2), and
primers using a KST mix at position Y78 would provide a mix of mutants containing
many of the single mutations needed. Anchoring off of this random pBS2-OYE 2.6
Y78XKST plasmid mix, primers using a KST mix at position I113 provided a random mix
containing up to 16 of the double mutations of interest. Anchoring off OYE 2.6 Y78T,
and OYE 2.6 Y78V with OYE 2.6 I113XKST primers as well as anchoring off OYE 2.6
I113T and OYE 2.6 I113V with OYE 2.6 Y78XKST primers provided us with four random
mixes containing up to 16 more double mutations of interest. The final four threonine-
valine double mutants had to be made individually. In this way the number of PCR
reactions, and the subsequent cloning steps, was reduced. Mutants obtained from both
the pooled KST randomized PCR cloning, and the completed set of valine and
threonine double mutants were used to assemble a random library containing small
residues at both the Y78 and the I113 position.
67
The three Baylis-Hillman substrates were used to screen the mutants in the OYE
2.6 Y78Xsm / I113Xsm randomized library. The positions that gave the most notable
results were then sequenced to determine the mutations present. By sequencing the
mutants after screening, we save time and effort by not sequencing numerous less
successful mutants. The majority of successful mutants were double mutants that were
successfully cloned. However, in a few cases promising positions were revealed to
contain concatomeric repeats of primer inserts in the sequence at the Y78 position.
Figure 2-8 shows the alignment of OYE 2.6 sequence with a sample containing portions
of primer inserts in the sequence. No concatomeric repeats of the primer was observed
in samples in which the I113 was targeted for cloning.
Positions in the OYE 2.6 Y78Xsm / I113Xsm randomized library that produced the
(R)-5 product were sequenced. Sequencing revealed that a cysteine mutation at
position I113 was present in most of the successful mutants. These substrates had
been extensively screened against an OYE 2.6 I113C single mutant and an OYE 2.6
Y78W / I113C double mutant in previous work.57 In fact, during the ISM experiments
anchoring off OYE 2.6 Y78W in which position I113 was randomized, it was revealed
that OYE 2.6 Y78W / I113C was the best double mutant in that library. OYE 2.6 I113C
and OYE 2.6 Y78W / I113C gave 100% and 98% conversion and 81% and 60% ee (S)
for the (S)-5 product of 2 respectively (Figure 2-9). However, the OYE 2.6 Y78Xsm /
I113C mutants had performed better than the single OYE 2.6 I113 and the OYE 2.6 Y78
/ I113 double mutant variants from the ISM experiments. Since these experiments,
which included a small amino acid mutation at position Y78, provided improved results
for obtaining the (R)-5 product from 2, we decided to fully explore the potential of double
68
mutants at these two positions. Thus, a complete library was assembled containing a
full matrix of all 36 double mutants.
OYE 2.6 Y78Xsm / I113Xsm Presequenced Library
Given the promising results of some of the mutants in the OYE 2.6 Y78Xsm /
I113Xsm randomized library, efforts were made to make all the small amino acid double
mutants of OYE 2.6 at positions Y78 and I113. Though the best results obtained from
screening the OYE 2.6 Y78Xsm / I113Xsm randomized library all contained the I113C
mutation, it was a concern that if we solely focused on the OYE 2.6 I113C double
mutants we might miss variants with remarkable results. Therefore, all 36 double
mutants would be made and screened. Substrate 2 was screened against the mutants
of the completed OYE 2.6 Y78Xsm / I113Xsm presequenced. Most of the mutants present
provided excellent conversion for 2. Figure 2-10 shows the results of screening the OYE
2.6 Y78Xsm / I113Xsm presequenced library against 2. However, the only set of mutants
that gave any (R)-5 product were the I113C mutants. Every OYE 2.6 I113C mutant gave
at least some (R)-5 product. Though no mutation at position Y78 stood out as the clear
choice for obtaining better yields of (R)-5, I113C was clearly the best substitution at that
position. At this point we felt there was not much more that could be done with solely
looking at these two positions. Moving forward, we chose to target a position that
worked well in combination with other double mutants made on the east side of the
active site, position F247.
OYE 2.6 Y78Xsm / I113C / F247X Randomized Libraries
We then investigated the west side of the OYE 2.6 active site, anchoring off the
best the double mutants discovered while screening the presequenced OYE 2.6 Y78Xsm
/ I113Xsm library. Previous mutagenesis projects57 revealed that a set of OYE 2.6 Y78W
69
/ I113C / F247A and F247H triple mutants would give good results for 2. Also, double
mutants that included a F247 mutation were in fact the best double mutants discovered
in the ISM project for obtaining (R)-5. Anchoring off of OYE 2.6 Y78A, Y78C, Y78G,
Y78S, Y78T, and Y78V / I113C, a set of 6 libraries were made in which position F247
was randomized with a set of primers containing a NNK mix of bases at the codon for
F247. A NNK mix contains 32 codons which include at least one codon for every
residue. This is the approach our group used during the ISM experiments to make
mutagenic libraries. Pooled samples of transformants were sequenced to access their
degeneracy using an evaluation method developed in our group during the ISM
experiments.26 Samples with sufficient degeneracy were further cloned into expression
strains and assembled into a randomized library. Table 2-3 contains all the Q scores
and the estimated number of amino acids obtainable from transformation with that
plasmid mix. Substrate 2 was used to screen the six OYE 2.6 Y78Xsm / I113C / F247X
libraries (Figure 2-11 through 2-16 shows the results of triple mutant screenings).
Unfortunately, few of the triple mutant variants gave any significant improvement over
the double-mutant anchor used to make the library. The six best results were
sequenced and not surprisingly four were the double mutant controls. The only two
triple mutants discovered during sequencing were OYE 2.6 Y78C / I113C / F247H and
F247W. With near 100% conversion and racemic ee, these mutations are the best
results discovered during this project and best variants of OYE 2.6 for obtaining (R)-5
from substrate 2 (Figure 2-9).
70
Experimental
General
Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained
from Integrated DNA Technologies (IDT). All other reagents were obtained from
commercial suppliers and used as received. Plasmids were purified on small scales by
Wizard® minicolumns (Promega Life Sciences) and on large scales using CsCl density
gradient ultracentrifugation.29 DNA sequencing was carried out by the University of
Florida ICBR using capillary fluorescence methods using standard protocols. LB
medium contained 5 g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.
ZY medium contained 5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052
contained 25% glycerol, 2.5% glucose, and 10% a-lactose monohydrate. NPS x20
contained 66 g/L (NH4)2SO4, 136 g/L KH2PO4, and 142 g/L Na2HPO4.
Cloning
Construction of plasmids used to make the OYE 2.6
The plasmid used as a template to make the OYE 2.6 libraries was pBS2, a
pET21a (+) derivative containing the gene for OYE 2.6-GST fusion protein. It was made
by the combined efforts of Despina Bougioukou and Bradford Sullivan (Appendix E,
figure E-2).34
Construction of the mutants in the OYE 2.6 Y78Xsm / I113Xsm randomized library
Templates used to make the mutants in the OYE 2.6 Y78Xsm / I113Xsm
randomized library were pBS2 derivatives obtained from an OYE 2.6 I113
presequenced library assembled by Adam Walton.34,57 Primers containing a KST mix of
nucleic acids at the OYE 2.6 Y78 position were used to make the pBS2-OYE 2.6
71
Y78XKST (OYE 2.6 Y78A, Y78C, Y78G, and Y78S) single mutant plasmid mix. Using the
pBS2-OYE 2.6 Y78XKST plasmid mix as a template, primers containing a KST mix of
nucleic acids at the OYE 2.6 I113 position were used to make the remaining OYE 2.6
Y78XKST / I113XKST double mutant plasmid mix. Primers containing a codon to give the
OYE 2.6 Y78T and Y78V mutations were used to make the four OYE 2.6 Y78T and
Y78V / I113T and I113V double mutants. Primers containing a KST mix of nucleic acids
at the OYE 2.6 Y78 and I113 positions were used with the appropriate pBS2 double
mutant to make the OYE 2.6 Y78XKST / I113T, OYE 2.6 Y78XKST / I113V, OYE 2.6 Y78T
/ I113XKST, and OYE 2.6 Y78V / I113XKST double mutant mixes. All primers used in this
chapter are listed in Appendix A, Table A-2. PCR was performed using 0.5 µL of 18 ng/
µL template, 5 µL of both 5 mM forward and reverse mutagenic primers, 1 µL of 10 mM
dNTP mix, 10 µL of 5X HF Phusion® Hot start buffer, 28 µL of sterile water, and 0.5 µL
of 2 U/µL Phusion® Hot Start II DNA Polymerase for a total reaction volume of 50 µL.
PCR was performed using a MJ Mini® thermocycler from BioRad. PCR samples were
run with an initial denaturation step at 98°C for 30 s, then a subsequent 25 cycles of
denaturation at 98°C for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C
for 3 min 30 s, after which the reactions were completed with a final extension step at
72°C for 7 min 30 s.
All PCR samples were cleaned using Wizard® Plus SV Gel PCR Clean up kits by
Promega, using the manufacturer instructions. Samples were then incubated overnight
with two doses of 0.5 µL of 20 U/µL DpnI at 37°C to remove the parent template. DpnI is
a nuclease that targets hemi methylated DNA and as such is ideal for removing the
superfluous template. The first dose of DpnI was added immediately after PCR clean up
72
and the second was added after 4 hours of digestion. After DpnI digestion, samples
were cleaned using Wizard® Plus SV Gel PCR Clean up kits by Promega, using the
manufacturer instructions.
Following the PCR work up, PCR samples were transformed by electroporation
into ElectroTen-Blue® electrocompetent cells (ETB) using a Gene Pulser® from BioRad.
Electroporation was carried out with 4 µL of PCR sample and 50 µL of ETB cells under
2.5 kV. Electroporated samples were incubated in 600 µL of SOC medium at 37°C for 1
h. Cells were then plated onto LB-amp agar medium and grown at 37°C for 36 h. The
best results were obtained from ETB daughter cells grown from the commercial stock
on the same day as the transformation would take place. Granddaughter cells provided
fewer transformants. Transformant cells were then pooled and plasmid DNA extraction
was performed with Wizard® Plus minipreps DNA purification system by Promega, using
the manufacturer’s instructions. Pooled plasmid samples were then sequenced by ICBR
using Sanger sequencing. Electropherograms obtained from Sanger sequencing were
measured to estimate the samples degeneracy. Degeneracy could be gauged for
samples using a KST primer mix (figure 2-17) by the method developed by Adam
Walton and Bradford Sullivan.26 Pooled plasmid samples with sufficient degeneracy
were used to transform an expression strain, E. coli BL21 (DE3) Gold cells.
Transformation was done using electroporation under 2.5 kV with 4 µL of 10 ng/µL of
pooled plasmid with 80 µL of E. coli BL21 (DE3) Gold electrocompetent cells.
Electroporated samples were incubated in 600 µL of SOC media at 37°C for 45 min.
Cells were then plated onto LB-amp agar medium and grown at 37°C overnight.
Transformants were then assembled into two 96 well plates. Three transformants per
73
possible codon would be required to obtain a good representation for each possible
codon in a pooled plasmid sample. Therefore, twelve transformants were taken from
each transformation of PCR samples using KST primer mix to accommodate the four
codons possible in a single KST codon mix, and 48 transformants were taken from the
transformation of the PCR sample using a double KST primer mix. Transformants were
grown in 600 µL of LB-amp in a 96 well plate overnight to reach saturation. The library
was completed with the transfer of 120 µL of saturated cultures into a new 96 well plate
containing 30 µL of 80% glycerol which brought the final concentration of glycerol to
15%.
Construction of the mutants in the OYE 2.6 Y78Xsm-I113Xsm presequenced library
The primers used for constructing a presequenced OYE 2.6 Y78Xsm-I113Xsm
library were from IDT (Appendix A, Table A-2). The plasmids were pBS2 derivatives
obtained either from the OYE 2.6 Y78Xsm-I113Xsm randomized library or from the OYE
2.6 Y78 presequenced library. PCR was done using the same conditions mentioned for
the OYE 2.6 Y78Xsm-I113Xsm randomized library, as was the PCR workup and
transformation into ElectroTen-Blue® electrocompetent cells. Cells were plated onto LB-
amp agar plates and grown at 37°C for 36 h. Transformant cells were sequenced to
verify that both desired mutations were present at the ICBR complex using Sanger
sequencing.
Successful plasmid samples were then transformed into expression strain E. coli
BL21 (DE3) Gold cells using the same conditions mentioned for the OYE 2.6 Y78Xsm-
I113Xsm randomized library. After mutations were verified by sequencing, transformants
were assembled into a 96 well plate. Transformants were grown in 600 µL of LB-amp in
a 96 well plate overnight to reach saturation. The library was completed with the transfer
74
of 120 µL of saturated cultures into a new 96 well plate containing 30 µL of 80% glycerol
which brought the final concentration of glycerol to 15%.
Addressing concatomeric primer inserts
It was discovered that mutating the Y78 position was problematic. Using our
primer design and PCR method, it has been possible to produce mutations at position
Y78 on occasion, but far more often than not the PCR product contained concatomeric
primer inserts like the ones observed during the experiments with the OYE 2.6 Y78Xsm /
I113Xsm randomized library. It is also noteworthy to mention that randomized primers
(be they either KST or NNK) were far less likely to make concatomeric products than
the single mutant primers. Efforts were made to address the issue of concatomers at
position Y78. Neither altering the PCR program, varying the concentrations of the PCR
components, nor replacing any of the commercially purchased solutions was effective in
reducing the number of concatomeric PCR products. Multiple different stock solutions of
OYE 2.6 Y78 primer were purchased and never reduced the amount of concatomeric
products. As to the I113 position, no transformant ever sequenced contained multiple
primer inserts at that position. Therefore to work around this problem, OYE 2.6 Y78
single mutants already made were used as the templates for making the 36 double
mutants and as such, all PCR reactions used OYE 2.6 Y78 mutants as templates and
primers containing mutations at position I113 to make all the double mutants.
Construction of mutants in the OYE 2.6 Y78Xsm / I113C / F247X randomized libraries
Templates used to make the mutants in the OYE 2.6 Y78Xsm / I113C / F247X
randomized libraries were pBS2 derivatives obtained from the OYE 2.6 Y78Xsm /
I113Xsm presequenced library. Primers containing a NNK mix of nucleic acids at the
75
OYE 2.6 F247 position were used to make the OYE 2.6 Y78Xsm / I113C / F247X triple
mutants were from IDT (Appendix A, Table A-2). PCR was done using the same
conditions mentioned for the OYE 2.6 Y78Xsm / I113Xsm randomized library, as was the
PCR workup, the transformation into ElectroTen-Blue® electrocompetent cells and
purification of pooled plasmid samples. Degeneracy could be gauged for samples using
a NNK primer mix with the equation in Figure 1-11. Pooled plasmid samples with
sufficient degeneracy were used to transform an expression strain E. coli BL21 (DE3)
Gold cells. Three transformants per possible codon would be required to obtain a good
representation for each possible codon in a pooled plasmid sample. Therefore, 94
transformants were taken from each transformation of PCR sample using NNK primer
mix to accommodate the 32 codons possible in a NNK mix. Two wells were left
available for the OYE 2.6 Y78Xsm / I113C double mutant anchor and OYE 2.6 wt., which
were included in wells G12 and H12 respectively to be used as controls. Transformants
were grown in 600 µL of LB-amp in a 96 well plate overnight to reach saturation. The
library was completed with the transfer of 120 µL of saturated cultures into a new 96
well plate containing 30 µL of 80% glycerol was added to a final concentration of 15%.
Substrates
A list of substrates and products is shown if Figure 2-2.
Methyl 2-(hydroxymethyl)acrylate (1)
Methyl 2-(hydroxymethyl)acrylate was prepared in our lab by Bradford Sullivan28
using the method developed by Turki et al.32 Methyl 2-(hydroxymethyl)acrylate was
detected during screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m).
The temperature program used began with an initial temperature of 100°C for 12 min,
followed by an increase at 20°C/min to a temperature of 180°C at which the program
76
remained for 5 min (GC method is listed as AZW3.Met in Appendix D). Methyl 2-
(hydroxymethyl)acrylate eluted near 11.8 min. The reduced S product eluted near 10.7
min and the reduced R product near 11.3 min.
2-(Hydroxymethyl)-cyclohex-2-enone (2)
2-(Hydroxymethyl)-cyclohex-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Rezgui and El Gaied.31 2-(Hydroxymethyl)-
cyclohex-2-enone was detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm x 30 m). The temperature program began with an initial temperature
of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at
which the program remained for 5 min (GC method is listed as AZW2.Meth in Appendix
D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The reduced S product
eluted near 10.2 min and the reduced R product near 10.8 min.
2-(Hydroxymethyl)-cyclopent-2-enone (3)
2-(Hydroxymethyl)-cyclopent-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Kar and Argade.30 2-(Hydroxymethyl)-
cyclopent-2-enone can be detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm x 30 m). The temperature program used began with an initial
temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature
of 180°C at which the program remained for 5 min (GC method is listed as AZW2.Meth
in Appendix D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The
reduced S product eluted near 11.4 and the reduced R product near 10.2 min.
Screening Assay
E. coli BL21 (DE3) Gold cells harboring plasmids containing OYE mutants of
interest were grown in a 96 well plate containing 600 µL of LB media with 200 μg/mL
77
ampicillin (LB-amp). Cells were grown at 37°C with 250 rpm of agitation overnight. The
saturated cultures were then used to inoculate a larger 2 mL square bottom 96 well
plate. This larger square bottom plate contained 600 µL of an auto-induction medium.
The auto-induction medium contained a mix of ZY media, 50x5052, 20x NPS, and 200
µg/mL ampicillin.33 Cells were induced in an aeration case developed at the University
of Florida by the machine shop in the Department of Chemistry. Induction occurred at
37°C with 350 rpm of agitation overnight. The increased agitation is required for
induction to occur. Induced cells were then separated from the auto-induction medium
by centrifugation. Auto-induction medium was removed and induced pelleted cells were
then resuspended in 600 µL of a reaction mixture. The reaction mixtures contained 50
mM KPi buffer with 100 mM glucose and 15 mM of the substrate of interest at pH 7.0.
Reactions were run at room temperature with 250 rpm of agitation overnight. Reactions
were quenched by adding 500 µL of ethyl acetate. The organic phase was separated by
centrifugation and extracted for analysis by GC.
Conclusions
It was previously shown during the OYE 1 W116 site saturation project that
position W116 is a significant site to use to engineer this protein. Modification at this
position has allowed new products to be obtained from using OYE 1 as a catalyst. The
ISM project showed that combing mutations at position at either I113 or F247 with the
linchpin mutation of Y78W in OYE 2.6 would allow that enzyme to make new exciting
products. This project proved that positions I113 and Y78 have substantially more room
for manipulation than previously shown. Regardless of which pair of the 36 double
mutations screened, the enzyme was still able to convert substrate to product. Also, the
OYE 2.6 Y78Xsm-I113Xsm presequenced library discovered a new linchpin cysteine
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mutation at position I113. The I113C double mutants made in this project have better
conversion and provide better ee than the best triple and double mutants obtained from
the ISM project. And by targeting the west side of the OYE 2.6 active site we gained the
same additive benefits for our double mutants as were gained from the ISM project.
Also, the results of the OYE 2.6 Y78Xsm / I113C / F247X screening did imply that the
triple mutants that contained an Y78A, Y78C, or Y78V worked the best. These libraries
contained the most functional variants of the six libraries screened. The OYE 2.6 Y78C /
I113C / F247H and F247W triple mutants were the best mutants discovered during this
project, and the best variants discovered to date for using this enzyme to obtain the
desired (R)-5 product. Should this project be continued, a complete library of the F247
position that anchors off OYE 2.6 Y78C / I113C would need to be made and screened
against substrate 2. This screening could verify which mutant at that position is the best.
79
Table 2-1. List of the presequenced alkene reductase libraries made by the Stewart group.
Library Plasmid Description
Alkene Reductase pDJB5, pDB13, pDJB8, pDJB17, pDJB9, pDJB11, pDJB15, pDJB24, pDJB22, pDJB26, pDJB21, pDJB23, pDJB25, pDJB27, pDJB19, pDJB29
OYE 1, OYE 2, OYE 3, oye, OYEA, OYEB, NemA, ppNema, ppOYE, SeOYE, OPR1, OPR2, OPR3, Leopr, Ltb4dh, YNL134c
pDJB5 contains a T7 promoter, Kanr, and a GST-tag
All other plasmids contain T7 promoter, Ampr, and a GST tag
OYE 1 W116 pDJB5-W116X Non-tagged, GAL1/GAL10 promoter, Kanr
OYE 1 Y196 pET3b-OYE Non-tagged, T7 promoter, Ampr
OYE 2.6 Y78 pBS2 GST-tagged, T7 promoter, Ampr
excludes Y78C, Y78F, & Y78N
OYE 2.6 I113 pBS2 GST-tagged, T7 promoter, Ampr
OYE 2.6 Y193 pBS2 GST-tagged, T7 promoter, Ampr
OYE 2.6 Y78W / I113 pBS2 GST-tagged, T7 promoter, Ampr
OYE 2.6 Y78Xsm / I113Xsm
pBS2 GST-tagged, T7 promoter, Ampr
OYE 2.6 S388 pFB1 Non-tagged, T7 promoter, Ampr
OYE 3 W116 pRP4 Non-tagged, T7 promoter, Ampr
80
Table 2-2. OYE 2.6 best variants discovered during the OYE 2.6 ISM studies.
Substrate Best variant for normal binding product
Best variant for flipped binding product
1 % conv 99, % ee 99 (S) OYE 2.6 wt % conv 99, % ee 91 (R) OYE 2.6 Y78W / F247A
2 % conv 99, % ee 99 (S) OYE 2.6 wt % conv 43, % ee 37 (S) OYE 2.6 Y78W / F247A
3 % conv 99, % ee 95 (S) OYE 2.6 wt % conv 97, % ee 98 (R) OYE 2.6 Y78W / I113V / F247H
(S)-(+)-Carvone % conv 48, % de 93 OYE 1 wt % conv 90, % de 99 OYE W116V
(R)-(-)-Carvone % conv 98, % de 97 OYE 1 wt % conv 78, % de 55 OYE W116A
Table 2-3. Q scores for NNK randomized libraries.
Library Q score Estimated number of amino acids obtainable from a transformation using this plasmid mix
OYE 2.6 Y78A / I113C / F247X 0.46 14.88
OYE 2.6 Y78C / I113C / F247X 0.77 18.83
OYE 2.6 Y78G / I113C / F247X 0.75 18.52
OYE 2.6 Y78S / I113C / F247X 0.75 18.52
OYE 2.6 Y78T / I113C / F247X 0.63 16.89
OYE 2.6 Y78V / I113C / F247X 0.61 16.68
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Figure 2-1. Flipped binding mode. This figure shows a 6-membered ring substrate undergoing catalysis in a flipped binding mode in OYE 2.6 wt. The silhouette of the 6-membered ring shows a normal binding mode.23
82
Figure 2-2. List of Chapter 2 substrates.
83
Figure 2-3. (S)-(+)-carvone bound in a flipped binding mode to the active site of OYE 1 W116I. The isopropylene group of (S)-(+)-carvone (blue) extends into the space between the residues at positions W116I and Y82. In the active site of OYE 1 W116I (S)-(+)-carvone can make use of this space due to the smaller isoleucine residue (PDB ID 4GE8).24
Figure 2-4. Mechanism of OYE 2.6. This figure shows the mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 2.6 (violet). OYE 2.6 uses H188 and H191 as hydrogen bonding partners to lock the carbonyl into position.
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Figure 2-5. Diagram of the residues in the OYE 2.6 active site. All positions except L115, and Q248 were targeted during the ISM experiments. Positions Y78, I113, and F247 were also targeted during this project.57
Figure 2-6. Malonate bound in the active site of OYE 2.6 Y78W-I113C (PDB ID 4M5P).34
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Figure 2-7. Substrate 1 (pink) and 2 (green) modeled into the active of OYE 2.6 Y78W and OYE 2.6 wt. Bound 6 membered rings have difficulty maintaining a flipped binding mode in OYE 2.6 because they extend into the area around the Y78 positions. (PDB ID 4DF2 and 4QAI).34
OYE 2.6 GACAGATCCACTTTCCCAGGTACTTTGCTTATCACTGAAGCTACTTTTGTCTCTCCTCAA
Concatmoers GACGGATCCACTTTCCCAGGTGCTTTGCTTATCACTGAAGCTACTTTTGTCTCTCCTCAA
*** ***************** **************************************
OYE 2.6 GCCTCTGGTTATGAAGGTGCTGCTCCAG--------------------------------
Concatmoers GCCTCTGGTGCTGAAGGTGCTGCTCCAGGTAAGCCTCTGGCGGAGAAGGTGCTGCTCCAA
********* *****************
OYE 2.6 ----------------------------GTATTTGGACTGACAAGCACGCTAAAGCATGG
Concatmoers GCCTCTGGTGGTGAAGGTGCTGCTCCAGGTATTTGGACTGACAAGCACGCTAAAGCATGG
********************************
OYE 2.6 AAGGTTATTACTGATAAAGTTCATGCCAACGGTTCTTTCGTTTCAACCCAGTTGATTTTT
Concatmoers AAGGTTATTACTGATAAAGTTCATGCCAACGGTTCTTTCGTTTCAACCCAGTTGGTTTTT
****************************************************** *****
Figure 2-8. Sequence alignment of OYE 2.6 with a sample containing primer inserts. Mutation sections are shown in blue, sections with portions of the primer inserted are show in green and yellow.
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Figure 2-9. Best variants discovered during both the ISM project and this project using a small residue matrix for obtaining the (R)-5 product from substrate 2.The small residue matrix library revealed that anchoring off I113C provided the best double mutants on the east side of the active site. Mutating a third position (F247) on the west side of the active site improved conversion even further. The triple mutants discovered to date are the OYE 2.6 Y78C / I113C / F247H and F247W mutants.
87
Figure 2-10. OYE 2.6 Y78Xsm / I113Xsm library screening results for substrate 2. OYE 2.6 wt. is not shown but gave 100% conversion with 100% ee (S). The only double mutant combinations that provided any (R)-5 product were ones that used I113C mutants. And all I113 mutants gave some (R)-5 product, with The OYE 2.6 Y78C / I113C providing marginally the most.
88
Figure 2-11. OYE 2.6 Y78A / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78A / I113C, the anchor double mutant. Nearly a third of the positions in this library gave conversion, and over half of those gave comparable (R)-5 to the anchor double mutant. Unfortunately, the positions sequenced were revealed to be the anchored double mutant.
89
Figure 2-12. OYE 2.6 Y78C / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78C / I113C, the anchor double mutant. This library provided two of the best triple mutants with positions H4 and F5. Sequencing revealed that position F5 was an OYE 2.6 Y78C / I113C / F247H mutant and H4 was an OYE 2.6 Y78C / I113C / F247H mutant. These are the best variants discovered in this study, and the best discovered to date.
90
Figure 2-13. OYE 2.6 Y78G / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78G / I113C, the anchor double mutants. This library did not provide enough positions with sufficient conversion to warrant sequencing. Though this is not surprising since the OYE 2.6 Y78G / I113sm mutants provided the lowest conversions during the OYE 2.6 Y78sm / I113sm presequenced screening.
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Figure 2-14. OYE 2.6 Y78S / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78S / I113C, the anchor double mutants. This library did not provide enough positions with sufficient conversion to warrant sequencing.
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Figure 2-15. OYE 2.6 Y78T / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78T / I113C, the anchor double mutant. This library did not provide enough positions with sufficient conversion to warrant sequencing.
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Figure 2-16. OYE 2.6 Y78V / I113C / F247X screening results for substrate 2. H12 is OYE 2.6 wt. and G12 is OYE 2.6 Y78V / I113C, the anchor double mutant. Nearly a fourth of the positions in this library gave conversion, and nearly half of those gave comparable (R)-5 to the anchor double mutant. Unfortunately, the positions sequenced were revealed to be the anchored double mutant.
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Figure 2-17. Calculations for obtaining a Qscore of a pooled plasmid mix from a KST primer mix using data from a theoretical sequencing electropherogram. This figure shows a theoretical electropherogram with theoretical peak heights. a) The fraction of each peak height at a position over all peak heights at that position is shown on the left side of each peak. Peaks correspond to: blue/thymine, orange/guanine, green/adenine, purple/cytosine, and the dashed black represents perfect degeneracy. b) The peak heights of each base is used to estimate the amount of codons containing that base at that position. c) The sum of those estimates are used to obtain a Q value (QK or QS) for that position. d) The sum of the weighted QK and QS values is used to calculate the Qscore for the pooled mix. The Qscore approaches perfect degeneracy (equal amounts of bases at each position) as it approaches 1.0.26
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CHAPTER 3 IMPROVING THE THERMOSTABILITY OF OYE 2.6 THROUGH PROTEIN
ENGINEERING
Background
Improving Thermostability through Mutagenesis
Protein engineering is not exclusively used to alter enzyme chemistry; it can also
be used to improve enzyme thermostability.60–67 Several recent papers have reviewed
the methods used to improve the protein’s stability.59,68–73 This is due to the commonly
held belief that thermophilic proteins (thermostable proteins) are more stable than their
mesophilic (non-thermophilic) analaogs.59,74–76 The properties of naturally thermostable
proteins have become models for improving the stability of mesophilic proteins. One
notable property of naturally thermophilic proteins is that they tend to have very rigid
structures. This suggests that increasing the rigidity of a mesophilic protein might
increase its thermostability. One common way to identify less rigid positions in a protein
is to find residues with elevated B-factors (or B-values) in the crystal structure.77–79
Since these values are often used for gauging this promiscuity of atomic motion in a
crystal structure, they are also used to gauge a position’s flexibility and stability.
Targeting such positions for mutagenesis with subsequent screening for variants with
greater thermotabilities has become a standard method in protein engineering.
Iterative saturation mutagenesis (ISM) can be an ideal engineering strategy for
progressing through different variants toward a more thermostable protein. For
example, Reetz and coworkers successfully employed an ISM approach to improve the
thermostability of Bacilius subtilis lipase A.80 The most impressive example involved five
mutations that increased the enzyme’s T50 by nearly 50°C (T50 is the temperature at
which the enzyme’s conversion drops to 50% and is commonly used as the standard for
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evaluating thermostability in enzymes). This result was obtained from a project that
included ten 1st round libraries randomized by a NNK codon set. Positions that were
targeted were those with high B-factors in the crystal structure of lipase A. As part of
these efforts, the Reetz group developed a program called B-fitter to identify positions
with the highest B-factors in a protein crystal structures.
A directed evolution project aimed at increasing an enzyme’s substrate or
product range generally targets positions within the enzyme’s active site; by contrast
improving an enzyme’s thermostability often involves residues spread throughout the
protein structure and often on its exterior. Positions of interest are those believed to
impair the protein’s stability by their high local motion that could lead to global unfolding.
Residues with high B-factors in crystal structures are a logical starting point. In addition,
surface residues involved in subunit-subunit contacts within quaternary structure may
also play a large role in dictating protein thermostability.
In a study begun by Filip Boratynski in our group, we applied this approach to the
alkene reductase OYE 2.6 from Pichia stipitis. Three crystal structures of this enzyme
had been solved by a former group member (Yuri Pompeu), which allowed us to identify
promising residues by B-factor analysis. These efforts had two major goals. First, we
hoped to increase the thermostability of OYE 2.6 to make it more practically useful for
organic synthesis. Protein thermostability often correlates with organic solvent stability,
and the hydrophobic substrates of this enzyme act as organic solvents at the high
concentrations desirable for preparative synthesis. Our second goal was to test the
hypothesis that mutations of residues with high B-factors actually yielded proteins with
lessened motion at these positions. In other words, if protein thermostability has been
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improved, does the variant actually show a lower B-factor at that position? To the best
or our knowledge, while this is implied by the methodology’s rationale, it has never been
addressed experimentally.
Boratynski’s B-factor analysis of OYE 2.6 revealed that the C-terminal region had
significantly higher B-factors than the rest of the protein (Figures 3-1 and 3-2). He
therefore targeted the ten positions with the highest B-factors identified by the B-fitter
program for random mutagenesis using a NNK codon set (Figure 3-3). Each of the ten
1st generation libraries were screened at elevated temperatures to identify variants with
greater thermostability. From this study, the best were S388P and S388A, which
increased T50 by 3°C and 1°C respectively. In addition, both E389G and E389S
increased the T50 by 1°C. Boratynski created and screened a 2nd generation library
anchored by S388P and randomized at the adjacent E389. Unfortunately, no double
mutation with greater thermostability was discovered.
Project Summary
The somewhat disappointing results after preparing and screening ten 1st and
one 2nd generation libraries prompted us to pursue a more selective approach to
improving the thermostability of OYE 2.6. Boratynski had targeted the ten positions with
the highest B-factors and most of them were located on the C-terminal loop and thus in
the same area. We instead chose positions with unusually high B-factors compared with
their neighboring residues. We hypothesized that such positions showed high local
motion in an otherwise quiet region. This approach yielded four positions, which were
targeted for randomized mutagenesis (Figures 3-4 and 3-5). We also investigated
residues that appeared to be involved with the homodimerization of OYE 2.6. Five
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positions at the dimer interface of OYE 2.6 were randomized using a NNK codon set
(Figure 3-6).
Once created, all mutants were screened at elevated temperatures to identify the
most thermostable variants. The best from each library were subsequently purified for
more extensive characterization, with particular emphasis on determining their T50
values. Our hope was to identify one or more variants that could be combined with
others found earlier to create an OYE 2.6 mutant with significantly enhanced
thermostability.
Testing whether thermostable variants actually showed smaller B-factors
required solving X-ray structures. Our crystallization conditions were capricious and
many crystals had to be screened in order to find the few that diffracted well. We
therefore carried out a more extensive screening of crystallization conditions to identify
a more reproducible methodology. While these efforts failed to yield better crystallization
conditions, we were able to characterize one of our most thermostable OYE 2.6 variants
by X-ray crystallography.
Results and Discussion
Residues with High Local B-Factors
Three crystal structures of OYE 2.6 were available at the start of this project
(PDB ID: 3TJL, 3UPW, and 4DF2).34 We examined the B-factors of positions in these
structures and identified the twenty positions with the highest values. Ten had already
been targeted by Boratynski during this earlier study. These included the two terminal
positions (S2 and E405), two adjacent positions on the exterior of the protein (E298 and
E299), and six positions on the loop leading toward the C-terminus (M386, D387,
S388P, E389, E390, and V391). Examining the ten next highest B-factor values, we
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selected four residues that were flanked by sites with smaller B-factors. It was believed
that these positions may be causing local instability and if altered, may reduce global
instability throughout the enzyme. The positions selected were E41, D141, E145, and
K330. PCR was used with a NNK codon set to make individual saturation mutagenesis
libraries at each of the four positions. After successful PCR amplification and
subsequent transformation, plasmids from a pooled sample of transformants were
sequenced to estimate the degeneracy of the libraries at the targeted positions. Q
values were determined and the libraries were evaluated using the method developed
by Sullivan and Walton. Table 3-2 shows the Q values for all libraries made in this
project.26
Once libraries with adequate degeneracy had been obtained, plasmids were
used to transform the E. coli overexpression host and 95 randomly selected individual
clones were tested for the ability to reduce 2-methyl-cyclopenten-1-one, a good
substrate for the enzyme (substrate 1 in this chapter). Lysates of each library member
were heated for 15 min in a 47°C water bath prior to alkene reduction at room
temperature. This initial screening revealed twelve mutations that warranted more
careful investigation (Figure 3-7 through 3-16). These included E41S and E41P; D141E,
D141H, D141R and D141; E145A, E145G, E145M and E145T; K330D and K330T.
During the next stage of screening, protein concentrations were normalized and lysate
samples were subjected to a heat treatment at 42°C for 15 min (Figure 3-17). Eight of
the original twelve mutants gave results that suggested further study (E41S and E41P;
D141E, D141H and D141R; E145A, E145G and E145T). Lysates from these mutants
were tested for catalytic activity at room temperature after heat treatment along a
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temperature gradient to determine their T50 values (Figure 3-18 through 3-22). The only
variant that performed consistently better than wild type was the D141E mutant.
Dimer Interface Residues
After examining the OYE 2.6 crystal structure, we selected five positions that
appeared to be important to the protein’s quaternary structure. These positions all
contained hydrophobic residues and were located on the exterior of the protein at the
dimer interface between the two subunits of OYE 2.6 (I214, W244, L260, I311 and
F307). None of these five sites had significantly higher B-factor values than average.
Randomized libraries were made for each of these positions using a NNK codon library
as described above. After obtaining libraries with adequate Q scores from sequencing a
pooled plasmid sample, plasmids were used to transform the E. coli overexpression
host and 95 randomly-chosen clones were screened as before (Table 3-2). The only
mutant discovered from these efforts that warranted further investigation was the I311L
variant. Unfortunately, this mutant was not consistently better than wild-type OYE 2.6
and it was thus not pursued further (Figure 3-17). In summary, no mutant with greater
thermostability was discovered by targeting the dimer interface for mutagenesis.
Combining Thermostabilizing Mutations
After examining 19 individual positions in OYE 2.6 using saturation mutagenesis,
the next step was to combine the most beneficial changes with the goal of additivity or
ideally, synergism. Boratynski observed that changes to S388 yielded the greatest
increase in thermostability. Pro and Ala proved the best replacements. One problem
with this study was that three variants were not included in the original library from the
original PCR amplification. To ensure that all replacements for S388 were examined, we
used site-directed mutagenesis to add these “missing” variants to the collection (S388I,
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S388N and S388Y). Progressive screening (small scale, large scale, and heat treatment
gradient temperature assays) confirmed that the S388A and S388P mutants were
optimal for thermostability at that position (Figure 3-18).
Based on all previous work, the two best mutations for enhancing the
thermostability of OYE 2.6 were D141E and S388P. We therefore prepared the double
mutant and tested the protein using the heat treatment gradient temperature assay. The
double mutant was additively more thermostable when compared to the two parent
single mutants, yielding a T50 value of 44ºC (Figure 3-22). This is 3ºC higher than the
wild-type and represents the most thermostable OYE 2.6 variant that has been
reported.
Crystallization of OYE 2.6 D141E-S388P
As noted previously, the original crystallization conditions for OYE 2.6 have not
been as reproducible as desired. This requires setting up multiple redundant crystal
trials of the same protein under the same conditions to obtain diffraction-quality crystals.
It also requires screening multiple crystals from the same well to identify one that yields
high-resolution data. For this reason, Boratynski was unable to obtain crystallographic
data for the S388P single mutant of OYE 2.6.
In his initial studies, Yuri Pompeu found three sets of promising conditions in the
Qiagen Classics I and II kits. We systematically varied pH, protein concentration,
precipitant concentration and addition concentration around these three initial hits.
Optimizing around the conditions in well 71 (Qiagen Classics I Suite) involved a matrix
of precipitant concentrations (30%, 20%, 10%, 5% (w/v) PEG 8,000 MME), pH values
(6.0, 6.5, 7.0), and protein concentration (5, 10, 20, 40 mg/mL) in 0.1 M sodium
cacodylate, 0.2 M sodium acetate supplemented with 3% isopropanol. A separate study
102
to optimize around conditions in well 91 (Qiagen Classics I Suite) used a matrix of
precipitant concentrations (30%, 20%, 10%, 5%, (w/v) PEG 5,000 MME), pH values
(6.0, 6.5, 7.0) and protein concentrations (5, 10, 20, 40 mg/mL) in 0.1 M Mes hydrate,
0.2 M (NH4)2SO4 with 3% isopropanol. Lastly, optimization around the original
conditions of well 27 (Qiagen Classics Suite II) utilized a matrix of precipitant
concentrations (3.5, 3.0, 2.4, 1.2, 0.6 M Malonate), pH values (6.0, 6.5, 7.0), protein
concentrations (5, 10, 20, 30, 40 mg/mL) and additive (4%, 3%, 2%, 1% isopropanol).
To our disappointment, none of these variations improved OYE 2.6
crystallization. Instead, in all cases these crystals would inevitably precipitate out of
solution as insoluble protein. Protein concentration was the only variable that had a
significant impact. Concentrations approaching 40 mg/mL provided fewer, larger
crystals with lower concentrations (ca. 10 mg/mL) afforded multiple, smaller crystals.
Though larger crystals obtained from the higher protein concentrations are of course
more desirable, they were often accompanied by a brown, insoluble protein precipitate.
Lower protein concentrations provided cleaner crystallization. One advance was that we
could use the hanging drop method to crystallize OYE 2.6; previously, only sitting drop
crystallization had been successful. This allowed us to obtain large numbers of crystals
for OYE 2.6 D141E / S388P.
We were able to obtain a high-resolution data set for the D141E / S388P mutant
to a resolution of 1.8 Å. Table 1 information for this structure can be found in Table 3-1.
After processing the data and solving the structure, we examined the C-terminal region
with particular interest. Proline substitution at position 388 did not appear to cause any
major structural perturbations to the C-terminal region. Our current working theory for
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the effect of the S388P mutation is that the more rigid proline may help lock the tail of
protein and thereby disfavor unfolding. Likewise, the D141E substitution did not impact
the structure significantly.
As noted previously, one key goal of this study was to determine when
successful mutations at positions selected for their high B-factors actually lower the B-
factors in the evolved protein. Our results were mixed. At position 388, the Pro residue
did indeed have a lower relative B-factor when compared to the wild-type Ser. On the
other hand, both the Asp and Glu residues at position 141 show essentially the same
relative B-factors, although the latter confers a 1ºC increase in T50.
The relative B-factor is the fraction of a position’s B-factor divided by the average
value of B-factors over the entire protein sequence. The relative B-factors of OYE 2.6
are highest near the C-terminus in all three of the previously-published structures
(Figure 3-23 through 3-28). In the structure of the D141E / S388P mutant, this peak in
the distribution of relative B-factors becomes a doublet with an indention at position 388.
In fact, using a normal distribution, the relative B-factor for position Ser 388 in OYE 2.6
wt. is 6 standard deviations greater than the mean of all relative B-factors (Figure 3-29).
By contrast, a Pro at position 388 has a relative B-factor to within 4 standard deviations
away from mean of all relative B-factors (Figure 3-30). While this remains a high local
value, it is clear that the mutation had a significant impact. For position 141 however,
this was not the case. The D141E mutation gave a slight improvement to OYE 2.6
thermostability, but did not significantly change the B-factor at that position. Using a
normal distribution, the relative B-factor for Asp at position 141 in wild-type OYE 2.6 is 2
standard deviations greater than the mean of all relative B-factors. A Glu at this position
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did not move the relative B-factor any closer towards the mean. It is tempting to
speculate that this lesser effect on B-factors is related to the mutation’s extremely
modest increase in T50 (1ºC).
Experimental
General
Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained
from Integrated DNA Technologies. All other reagents were obtained from commercial
suppliers and used as received. Plasmids were purified on small scales by Wizard®
minicolumns (Promega Life Sciences) and on large scales using CsCl density gradient
ultracentrifugation. DNA sequencing was carried out by the University of Florida ICBR
using capillary fluorescence methods using standard protocols. LB medium contained 5
g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl. ZY medium contained
5 g/L Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052 contained 25% glycerol,
2.5% glucose, and 10% a-lactose monohydrate. NPS x20 contained 66 g/L (NH4)2SO4,
136 g/L KH2PO4, and 142 g/L Na2HPO4.
Cloning
Construction of the plasmid used as a template for the OYE 2.6 thermal stability libraries
The plasmid used as a template to make all OYE 2.6 site saturation mutagenesis
libraries was pFB1 (Appendix E, Figure E-3), a pET22b derivative containing the gene
for wild-type OYE 2.6 with no extraneous affinity tags. It was made by the combined
efforts of Filip Boratynski and Bradford Sullivan. CsCl-purified pET22b and pBS2 were
digested with both NdeI and XhoI and the desired fragments were purified by low melt
105
agarose gel electrophoresis. The two fragments were joined by T4 ligase and the
desired plasmid (pFB1) was identified after transformation into electro competent E. coli
cells.
Construction of OYE 2.6 libraries
Primers were designed to replace individual codons with a NNK mix (Appendix A,
Table A-3). This was carried out for E41, D141, E145, K330, I214, W244, L260, I311,
and F307. PCR was performed using 0.5 μL of 18 ng/μL pFB1 template, 5 μL of both 5
mM forward and reverse mutagenic primers, 1 μL of 10 mM dNTP mix, 10 μL of 5X HF
Phusion® Hot start buffer, 28 μL of sterile water, and 0.5 μL of 2 U/μL Phusion® Hot
Start II DNA Polymerase for a total reaction volume of 50 μL. PCR was performed using
a MJ Mini® thermocycler from BioRad. PCR amplifications were run with an initial
denaturation step at 98°C for 30 s, then a subsequent 25 cycles of denaturation at 98°C
for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C for 3 min 30 s, after
which the reactions were completed with a final extension step at 72°C for 7 min 30 s.
All PCR samples were cleaned using Wizard® Plus SV Gel PCR Clean up kits by
Promega, using the manufacturer instructions. Samples were then incubated overnight
with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent template. DpnI
selectively targets hemi methylated DNA and as such is ideal for removing any
remaining template DNA from the PCR amplifications. The first aliquot of DpnI was
added immediately after PCR clean up and the second after 4 hours of digestion. After
DpnI digestion, samples were purified using Wizard® Plus SV Gel PCR Clean up kits by
Promega, using the manufacturer instructions.
The DNA samples were used to transform ElectroTen-Blue® electrocompetent
cells (ETB) using a BioRad Gene Pulser®. Electroporation was carried out with 4 μL of
106
DNA sample and 50 μL of ETB cells at 2.5 kV. Electroporated samples were
immediately incubated in 600 μL of SOC medium at 37°C for 1 h. Cells were then plated
onto LB-amp agar medium (200 μg/mL ampicillin) and grown at 37°C for 36 h. The best
results were obtained from ETB daughter cells grown from the commercial stock on the
same day as the transformation would take place. Granddaughter cells provided fewer
transformants. Transformants were pooled and plasmid DNA extraction was performed
with Wizard® Plus minipreps DNA purification system by Promega, using the
manufacturer instructions. Pooled plasmid samples were then sequenced by ICBR
using Sanger sequencing. Electropherograms obtained from Sanger sequencing were
measured to estimate the samples degeneracy. Degeneracy could be gauged for
samples using a NNK primer mix with the equation in Figure 1-11 in Chapter 1. Pooled
plasmid samples with sufficient degeneracy were used to transform an expression strain
E. coli BL21 (DE3) Gold cells. To ensure complete coverage of the 32 possible codons
present in a NNK library, 95 randomly-chosen transformants were selected.
Transformants were grown in 600 μL of LB-amp in a 96 well plate overnight to reach
saturation along with the wild-type OYE 2.6 control. From these pre-cultures, 120 μL
was transferred into a new 96 well plate containing 30 μL of 80% glycerol. This brought
the final concentration of glycerol to 15% which is sufficient for cryoprotection of cells at
-80°C.
Substrates
2-Methyl-2-cyclopenten-1-one (1)
2-Methyl-2-cyclopenten-1-one was purchased from Sigma Aldrich. 2-Methyl-2-
cyclopenten-1-one can be detected during screening by GC-FID using a DB-17 column
(0.25 mm x 30 m). The temperature program used began with an initial temperature of
107
60°C for 5 min, followed by an increase at 20°C/min to a temperature of 200°C at which
the program remained for 8 min. (GC method is listed as FB1.Meth in Appendix D). 2-
Methyl-2-cyclopenten-1-one eluted near 6.5 min. The reduced product eluted near 4.2
min. The chirality of the reduction product was not of interest to this project and thus
was not determined.
Screening
E. coli BL21 (DE3) Gold cells harboring pFB1 derivatives of interest were grown
in a 96 well plate containing 600 μL of LB-amp medium. Cells were grown at 37°C with
250 rpm of agitation overnight. The saturated cultures were then used to inoculate a
larger 2 mL square bottom 96 well plate. This larger square bottom plate contained 600
μL of ZYP-5052 auto-induction medium. This auto-induction medium contained a mix of
ZY medium, 50x5052 solution, 20x NPS solution, and 200 μg/mL ampicillin.81 Cells
were induced in an aeration case developed at the University of Florida by the machine
shop in the Department of Chemistry. Induction occurred at 37°C with 350 rpm of
agitation overnight. The increased agitation is required for induction to occur. Induced
cells were then separated from the auto-induction medium by centrifugation at 6°C for
10 min using 1,500 × g. Auto-induction medium was removed and cells were lysed
using freeze-thaw method. This involved freezing the plate at -80°C for 1 h followed by
thawing at room temperature for 30 min. After three rounds of freeze-thaw lysis, cells
were resuspended in 250 μL of lysis buffer containing 2 mg/mL of lysozyme and 100
mM KPi at pH 7.0. Lysis continued with an incubation in 37°C with 250 rpm agitation for
2 h. The crude lysates were heat-treated by incubating in a hot water bath at 47°C for
15 min. This temperature and time was selected because it was near the threshold for
inactivation of wild-type OYE 2.6 in this assay. An aliquot (200 μL) of heat treated cell
108
lysate was transferred to a new plate containing 250 µL of reaction mixture to determine
the remaining alkene reductase activity. The final assay concentrations were 5 mM
alkene substrate 1, 200 mM glucose, 44 U GDH, 0.3 mM NADP+, 100 mM KPi buffer at
pH 7.0. Reactions were run at 37°C with 250 rpm agitation for 3 h. Reactions were then
quenched by adding 500 μL of ethyl acetate. The organic phase was separated by
centrifugation and extracted for analysis by GC.
Large scale screening was performed on selected mutants of particular interest.
E. coli BL21 (DE3) Gold cells harboring pFB1 derivatives encoding OYE 2.6 mutants
were taken from streaks grown on a LB-amp agar plate. Cells were grown in 1 mL of
LB-amp for at least 6 h at 37°C with 250 rpm of agitation to reach saturation. After
saturation, 0.5 mL of inoculum was transferred to 30 mL of ZYP-5052 auto-induction
medium and induced overnight at 37°C with 350 rpm of agitation. After auto-induction
was complete, the cell cultures were pelleted by centrifugation, resuspended in 2 mL of
100 mM KPi buffer at pH 7.0, and lysed with two passes through a French Press. The
lysate was clarified by centrifugation and the pelleted debris was discarded. A Bradford
assay was used to determine the protein concentration of the supernatant. All mutant
protein samples were adjusted to 300 µg/mL by diluting with 100 mM KPi. An aliquot (50
μL) of the normalized protein solution was transferred to 250 μL of a reaction mixture
which had been preheated to 42°C using a thermocycler for 30 min. This temperature
was selected because it was at or slightly higher than the T50 of wild-type OYE 2.6. The
reaction mixtures contained 5 mM alkene substrate 1, 200 mM glucose, 44 U of GDH,
0.3 mM NADP+, and 100 mM KPi buffer at pH 7.0. The regeneration system used to
make NADPH which reduces the FMN of OYE and allows the protein to turnover is
109
shown in Figure 3-31. The heat treatment lasted 15 min after which the reactions were
removed from the thermocycler. Reactions were run at 37°C with 250 rpm agitation for
90 min, then quenched by adding 300 μL of ethyl acetate. The organic and aqueous
phases were separated by centrifugation and the organic phase was analyzed by GC-
FID.
OYE 2.6 mutants that converted as much or more substrate than wild-type OYE
2.6 were further evaluated using a temperature gradient assay. During this assay, cells
were grown, induced, lysed and normalized using the same method for the large scale
screening. Once the protein concentrations of all samples had been determined and
normalized to a common concentration, evaluation by heat treatment followed. The
reaction buffer mixture was preheated to the heat treatment temperature using a
thermocycler for 30 min. An aliquot (50 µL) of normalized protein solution was
transferred to 125 µL of a preheated reaction buffer mixture. Protein samples were heat
treated for 15 min before being removed from the thermocycler. 125 µL of reaction
mixture was added to each post heat treatment sample bringing the volume to 300 μL.
The now completed reaction mixtures contained 5 mM alkene substrate 1, 200 mM
glucose, 44 U of GDH, 0.3 mM NADP+, and 100 mM KPi buffer at pH 7.0. Reactions
were run at 37°C with 250 rpm agitation for 90 min. Reactions were quenched by
adding 300 μL of ethyl acetate, then the organic and aqueous phases were separated
by centrifugation and the organic phase was analyzed by GC-FID.
Protein Purification and Crystallization of OYE 2.6 Mutants
Protein purification for OYE 2.6 mutants was carried out using the procedure for
purifying the OYE 2.6 protein developed previously in our lab,34 which is a modification
of the procedure originally developed by Massey for purifying Saccharomyces
110
pastorianus OYE 1.6 E. coli BL21 (DE3) Gold cells harboring a derivative of pFB1
encoding the desired mutant were grown at 37°C in a 4 L New Brunswick Scientific M19
fermenter containing LB-amp. Cells were grown in the fermenter with 600 rpm of
agitation for 2 h to achieve mid log phase. Cells were induced by adding IPTG and
glucose to final concentrations of 0.4 mM and 100 mM, respectively. The culture was
grown at 30°C with 600 rpm of agitation for an additional 4 h. The culture was chilled at
4°C for 30 min before centrifugation at 5,000 g. Wet cell pellets were then resuspended
in 100 mM Tris-Cl buffer containing 10 μM PMSF at pH 8.0. Cells were then lysed under
12,000 psi with the aid of a French Press. Lysates were centrifuged at 15,000 g for 1 h.
Nucleotides were precipitated by adding protamine sulfate to a final concentration of 1
mg/mL and stirring at 4°C for 20 min. The supernatant was separated by centrifugation
at 15,000 g for 20 min, then protein was precipitated by adding 5 partitions of
ammonium sulfate every 5 min to achieve a final concentration of 78% saturation. The
protein precipitate was separated by centrifugation at 15,000 g for 1 h.
Purification of OYE 2.6 by an N-(4-hydroxybenzoyl) aminohexyl agarose affinity
column requires that the active site to be emptied of any bound ligand that would
interfere with binding to the phenol moiety of the column matrix. This was accomplished
by successive buffer exchanges during dialysis. The ammonium sulfate pellet obtained
from the salt cut was resuspended in 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF
buffer at pH 8.0. This was dialyzed against 1 L of 100 mM Tris-Cl, 100 mM (NH4)2SO4,
10 μM PMSF buffer at pH 8.0 overnight at 4ºC. The following day, the sample was then
dialyzed against 1 L of buffer containing 10 mM sodium dithionite for 2 h at 4ºC. After 2
h, the buffer was exchanged for a fresh 1 L of buffer containing 10 mM sodium dithionite
111
and dialysis continued for 2 h. The sample was then transferred to a fresh 1 L of buffer
without dithionite and dialyzed for 2 h, after which, the buffer was exchanged with a final
1 L of fresh buffer and dialyzed overnight. The final sample was then centrifuged at
15,000 g for 30 min to remove any insoluble debris accumulated during dialysis.
The affinity column was packed with 3 mL of the matrix and was equilibrated with
100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF buffer at pH 8.0, prior to use. The
flow rate during purification was 0.5 mL/min. Elution of non-bound protein was
monitored by absorbance at 280 nm and a baseline established prior to loading protein
sample onto the column. Dialyzed protein samples were loaded onto the affinity column
in 10 mL portions. Binding of OYE 2.6 turned the column greenish brown. Bound OYE
2.6 was washed with starting buffer until 280 nm absorbance returned to the baseline;
this required anywhere between 30-60 mL of buffer. Protein was eluted by washing with
100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF, 4 mM sodium dithionite at pH 8.0.
OYE 2.6 was then further purified by gel filtration with a Superdex 200 column
(Pharmacia) using 50 mM Tris-Cl, 50 mM NaCl buffer at pH 7.5. Pooled fractions
containing the desired protein were then concentrated by ultrafiltration using an Amicon
centrifugation tube to a final concentration of 40 mg/mL. Protein concentration was
determined by absorbance at 280 nm using an extinction coefficient (ε) and molecular
weights (MW) estimated by protparam (OYE 2.6 had an ε of 55,810 M-1cm-1 with a MW
of 45,274 Da).35
Crystals were grown using the published conditions.34 Wells contained 6 μL of
crystallization solution combined with 6 μL of 40 mg/mL protein in 50 mM Tris-Cl, 50
mM NaCl buffer pH 7.5 and used hanging drop vapor diffusion. The crystallization
112
solution contained 2.4 M malonate with 3% isopropanol at pH 7.0. The best crystals
obtained were grown in 12 days at 6°C. Crystals were soaked in a harvesting buffer of
2.4 M malonate at pH 7.0, 3% isopropanol with 15% (v/v) glycerol before being flash
cooled in liquid nitrogen and sent for data collection.
Data Collection and Structure Solution of OYE 2.6 D141E-S388P
The best crystals diffracted to a maximum usable resolution of 1.81 Å using the
X6A beamline at Brookhaven National Laboratory. The unit cell measured was 126.93
126.93 122.71 90 90 120 Å and the crystals belonged to space group P 63 2 2. The
asymmetric unit contained 1 molecule, a solvent content of 60.95%, and a Matthew’s
coefficient of 3.15 Å3/Dal.36
Reflection data were processed using the iMOSFLM program in the CCP4
program suite to a resolution of 1.81 Å.37 Phases were obtained using the Phaser-MR
utility of the PHENIX program suite by molecular replacement using a modification of P.
stipitis OYE 2.6 (PDB code 3TJL) as the search model.38 All ligands and water
molecules were removed prior to molecular replacement. Inspection of the model
showed one OYE 2.6 chain present in the asymmetric unit. The best solution for the
space group was determined to be P 63 2 2. The initial model was well defined
throughout the scaffold of the protein giving an Rfree value of 0.27. Further refinement
using the xyz coordinates, B-factors, real-space, and occupancies refinement strategy
features in PHENIX refine, as well as continued cycles of model building using the
structure validation tools in COOT, produced a model with an Rfree of 0.19. Once the
protein scaffold was established, the density in the OYE 2.6 active site was addressed.
Malonate from the crystallization buffer was present in the active site of most of the
OYE 2.6 structures and the D141E / S388P variant of OYE 2.6 was no exception.
113
Malonate fit the active site density well and was modeled into the active site as well as
additional areas throughout the model providing an Rfree of 0.18. The FMN cofactor was
modeled into the structure and subsequent refinement provided an Rfree of 0.17. This
was followed by further model work-up and refinement providing a final Rfree of 0.17.
B-Factor Data and Statistics
B-factors were evaluated as B-factor fractions. B-factors fractions were
calculated as the average of B-factors for all atoms of a position over the average of all
B-factors for every position. B-factors were determined to be significantly different by a
normal distribution test. Aspartate at position 141 in wild-type OYE 2.6 had a B-factor
fraction of 2.0, which was 3 standard deviations outside the average B-factor fraction of
all positions in the wild-type OYE 2.6 structure. A Glu at position 141 had a B-factor
fraction of 2.5, which remained 3 standard deviations outside the average B-factor
fraction of all positions in the D141E / S388P OYE 2.6 structure. This means that the
glutamate mutation did not significantly alter the B-factors at position 141. Position S388
had B-factor fraction of 2.9 in the wild-type OYE 2.6 structures, which was 6 standard
deviations outside the average B-factor fraction of all positions in the wild-type OYE 2.6
structure. The Pro at position 388 had a B-factor fraction of 3.9 which moved that
position to 4 standard deviations outside the average B-factor fraction of all positions in
the D141E / S388P OYE 2.6 structure. This means that the S388P mutation significantly
decreased the B-factor value at that position.
Conclusions
In this series of projects, we have discovered five mutations at three positions
that have consistently improved the thermostability of OYE 2.6 (D141E, S388A, S388P
E389G and E389S). Only the D141E / S338P OYE 2.6 double mutation was successful
114
at combining two positions for an additive effect. The combination of these two
mutations increased the T50 of OYE 2.6 between to 3°C, which was additive if not
synergistic. While these results are much more modest than those of Reetz, who
increased the thermostability of lipase A by 50°C, we have been able to both
successfully increase our protein’s stability and corroborate our hypothesis with
crystallographic data (Figure 3-29 and 3-30).80 B-factor data obtained from our crystal
structure proved that by improving the thermostability at position S388, we could
decrease the B-factor at that position relative to the protein. Whether there is a
correlation between the minor increases in thermostability general protein stability is yet
to be definitively tested. However, observations made during crystallization of this
protein suggest that this is the case. As mentioned earlier, while OYE 2.6 and some
OYE 2.6 variants can be crystallized using the published conditions; however, this was
not consistent, particularly for the mutants. This inability to crystallize interesting OYE
2.6 variants clearly limits us from structural investigations. On the other hand, mutants
from this study, selected for enhanced thermostability, crystallized very well and
consistently. This suggests that the D141E / S338P double mutant could be used as a
vehicle for examining crystal structures of other mutants developed by our lab that
proved refractory to crystallography.
Future attempts to increase the thermostability of OYE 2.6 should use a different
approach to mutagenesis. To date, our group has targeted over 33 positions in OYE 2.6
for single site saturation mutagenesis libraries (8% of the entire protein). Over half of
those positions were probed for the purpose of improving thermostability. Covering this
amount of ground would traditionally be done with using a technique like cassette
115
mutagenesis. Though it is less precise than single site mutagenesis, this type of
technique allows for a broader range of positions to be targeted. Given that we have
exhausted the rational and even semi-rational positions, this would be the ideal
approach of mutagenesis to use to further improve the thermostability of OYE 2.6.
116
Table 3-1. Crystallographic data collection and refinement statistics
OYE 2.6 D141E-S388P
X-Ray Source X6A beamline
Brookhaven National
Laboratory
Space Group P 63 2 2
Unit Cell Dimensions
126.93 126.93 122.71
90 90 120
a = b, c (Å)
Resolution (Å) 38.88 - 1.798
(1.862 - 1.798)
Unique Reflections 53506 (5254)
Completeness (%) 1.00 (1.00)
Multiplicity 11.8 (11.5)
Rsym 0.116 (0.632)
I/σ (I) 15.7 (4.0)
Rwork 0.1454 (0.1832)
Rfree 0.1697 (0.2048)
Ramachandran
Statistics
Favored (%) 97
Allowed (%) 3.2
Outliers (%) 0
Number of Protein,
Solvent, and Ligand
Atoms
3220, 481, 55
Average B Factors
(Å2) 20.93
Protein 19.64
Ligands 21.50
Solvent 29.50
117
Table 3-2. Q scores for NNK randomized libraries.
Library Q score Estimated number of total amino acids obtainable from a transformation using this plasmid mix
OYE 2.6 E41X 0.78 18.95
OYE 2.6 D141X 0.71 18.02
OYE 2.6 E145X 0.86 20.00
OYE 2.6 K330X 0.56 15.99
OYE 2.6 I214X 0.65 17.19
OYE 2.6 W244X 0.68 17.57
OYE 2.6 L260X 0.65 17.19
OYE 2.6 F307X 0.73 18.33
OYE 2.6 I311X 0.76 18.67
118
Figure 3-1. The fraction of B-factors for each position over the average B-factors of all positions in the structure (B-factor fraction). The B-factors for each position are taken as an average of all atoms for that position obtained from the crystal structure. These atoms include both the residue atoms and the peptide backbone atoms for a position. Since the magnitudes of B-factors can differ across different structures, using a fraction that compares a position relative to the whole structure becomes more ideal. As the B-factor fraction of a position moves further and further above 1, it becomes easier to distinguish that position as one with a higher B-factor.
Figure 3-2. The relative B-factors of all three published OYE 2.6 wild type structures. The magnitude of B-factor are shown along a color gradient from blue with the lowest values, to yellow with higher values, to red with the highest values. Though the B-factor of a position may be different from structure to structure, the relative B-factor value for each position compared to the overall structure, is very consistent. If a position has a high B-factor value in one structure it will likely be high in all structures.
119
Figure 3-3. The positions targeted during the ISM thermostability project, their region in the protein, and their B-factors for structure 3TJL.
Figure 3-4. The positions targeted during the local maximum project, their region in the protein, and their B-factors for structure 3TJL.
120
Figure 3-5. The B-factor values for the positions targeted during the local maximum project. The B-factor values for positions target as well as those of the adjacent positions in three published structures of OYE 2.6 wt. These four positions were selected because they have higher B-factors than there adjacent positions and are considered local maxima of protein instability.
Figure 3-6. The positions selected for mutagenesis during the dimer interface project. OYE 2.6 is a homodimer and has a hydrophobic region connecting the two subunits. The top subunit is shown in purple while the second lower subunit is shown in orange. Leu 260 has two alternate conformations in the crystal structure.
3TJL 3UPWPDB Code
Position
L40
E41
22
38
B-factor
22 31
4DF2
D42
S140
A142
D141
K144
E145
A146
F329
T331
K330
42 53 55
27 28 36
33 34 41
34 35 40
48 53
38 44 51
25 25 35
32 30 37
25 26 36
21 23 33
32 39 51
121
Figure 3-7. The results for the small scale screening of the OYE 2.6 S388 library testing all 19 possible replacements with substrate 1.
Figure 3-8. The results of screening the OYE 2.6 E41X NNK randomized library.
122
Figure 3-9. The results of screening the OYE 2.6 D141X NNK randomized library.
Figure 3-10. The results of screening the OYE 2.6 E145X NNK randomized library.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
5
OYE2.6 E145X NNK randomized library
Conversion 0% 1- 25% 25-50% 50-75% 75-99%
Color
Total 91 0 0 0
123
Figure 3-11. The results of screening the OYE 2.6 K330X NNK randomized library.
Figure 3-12. The results of screening the OYE 2.6 I214X NNK randomized library.
124
Figure 3-13. The results of screening the OYE 2.6 W244X NNK randomized library.
Figure 3-14. The results of screening the OYE 2.6 L260X NNK randomized library.
125
Figure 3-15. The results of screening the OYE 2.6 F307X NNK randomized library.
Figure 3-16. The results of screening the OYE 2.6 I311X NNK randomized library.
126
Figure 3-17. The results from the large scale screening assay. The libraries with mutations that warranted a large scale screening were: OYE 2.6 D141X, E145X, I311X, K330X, & S388P. Many successful reactions from the small scale screening (Hits) were revealed to be wild type (wt. Hits) after sequencing. The results of the sequencing are also shown in the bottom right corner.
127
Figure 3-18. The results of the best mutants from all ten ISM-libraries.
Figure 3-19. The results of the best mutants from OYE 2.6 E41X.
128
Figure 3-20. The results of the best mutants from OYE 2.6 D141X.
Figure 3-21. The results of the best mutants from OYE 2.6 E145X.
129
Figure 3-22. The results of the best mutants from all projects.
130
Figure 3-23. The positions targeted in both the ISM thermostability (circled in red) and local maximum projects (circled in yellow). Both projects targeted positions based on their B-factors. The region of Helix 25 is by far the area with the highest B-factor fractions. The most successful position targeted was OYE 2.6 S388P and is located near the pinnacle of that peak.
Figure 3-24. The relative B-factor fractions from the OYE 2.6 D141E-S388P structure. The conserved mutation at position D141 to a glutamate reduced the T50 by 1°C. This mutation was only marginally successful and did not impact the structure enough to alter the relative B-factor fraction at that position (circled in yellow). The mutation at position S388 to a proline reduced the T50 between 2-4°C and lowered the B-factor fraction of that position (circled in red).
131
Figure 3-25. The relative B-factor fraction of all OYE 2.6 positions along the 3UPW structure.
Figure 3-26. The relative B-factor fraction of all OYE 2.6 positions along the 3TJL structure. The pattern of B-factor fractions obtained from this structure is near identical to that of the 3UPW.
132
Figure 3-27. The B-factor fractions for all three published OYE 2.6 wt. structures.
Figure 3-28. The B-factor fractions of both the three OYE 2.6 wt. structures (blue, red, and yellow) and the OYE 2.6 D141E-S388P structure (purple). Though there is greater variance in the magnitude of the double mutant B-factor fraction the overall pattern is consistent. One significant difference is the broad “singlet” peak at helix 25 for the three wild type structures has becomes a “doublet” for the double mutant structure due the indent caused by S388P lowering the B-factor fraction.
133
Figure 3-29. The relative B-factor fractions of OYE 2.6 wt. in structure 3TJL. The standard deviation of the mean (σ) is 0.36. Position D141 has a B-factor fraction of 2.0 and is within 3 σ of the mean of all B-factor fractions. Position S388 has a B-factor fraction of 2.9 and is within 6 σ of the mean of all B-factor fractions.
Figure 3-30. The relative B-factor fractions of OYE 2.6 D141E-S388P structure. The standard deviation of the mean (σ) is 0.77. Position D141 has a B-factor fraction of 2.5 and is within 3 σ of the mean of all B-factor fractions. Position S388P has a B-factor fraction of 3.9 and is within 4 σ of the mean of all B-factor fractions.
134
Figure 3-31. The regeneration system used to make NADPH which reduces the FMN of OYE and allows the protein to turnover.
135
CHAPTER 4 THE STRUCTURE OF Saccharomyces cerevisiae OLD YELLOW ENZYME 3
Background
Crystallization of OYE Family Members
The crystal structure of Old Yellow Enzyme 1 from Saccharomyces pastorianus
(OYE 1) solved by Karplus and Fox in 1994 provided the first structural information on a
member of the OYE family.20 This structure identified key positions responsible for
binding FMN within the active site. These include T37, G72, Q114, and R243, which act
as either hydrogen bond donors or acceptors and L36, which makes hydrophobic
contacts with the C7 and C8 methyl groups of FMN (Figure 4-1). The crystal structure
also provided insight into the critical residues that interact with bound substrates. H191
and N194 form hydrogen bonds with the carbonyl of the bound substrate, which locks it
into position within the active site. These hydrogen bonds also stabilize the oxyanion
intermediate prior to its protonation (Figure 4-2). Other important residues were also
identified in OYE 1 active site (Figure 4-3). Our group has extensively studied site-
directed mutants of W116.22,23 Several residues on loop 6 (P295, F296, and L297)
might also play important roles since this loop lies on the southwest side of the active
site and is believed to be responsible for opening the active site to accommodate
NADPH binding / NADP+ release during the catalytic cycle (Figure 4-4).
In addition to our extensive work with S. pastorianus OYE 1, our group has also
explored several other OYE 1 homologs. Pichia stipites OYE 2.6 was used for a large
directed evolution study, and its crystal structure was solved by Yuri Pompeu as part of
these efforts.34 This information improved our mutagenic planning and also allowed us
to observe the effects of our mutations had on the OYE 2.6 structure. The structure of
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OYE 2.6 showed very similar interactions between the protein and the FMN cofactor as
were observed previously for OYE 1 (Figure 4-1). In OYE 2.6, H188 and H191
appeared to be positioned to form hydrogen bonds with the substrate carbonyl (Figure
4-2). Other potentially important residues that might interact with the substrate
(analogous to those in OYE 1) were also revealed (Figures 4-3 and 4-4).
Old Yellow Enzyme 3 (OYE 3) is an OYE homolog from Saccharomyces
cerevisiae originally discovered by the Massey group during their efforts to clone the
gene encoding S. pastorianus OYE 1.9 OYE 3 shares significant sequence similarity to
OYE 1 (80%). We had included OYE 3 in our original collection of overexpressed OYE
1 homologs and this enzyme has also been explored by others (Figure 4-5).56 Our
collaborators (Professor Elisabetta Brenna) was interested in OYE 3 because it yielded
products with opposite stereoselectivites for some substrates when compared to OYE 1
and OYE 2.52 Because of the very high sequence identity between OYE 3 and OYE 1,
we decided to solve the crystal structure of the former to understand its divergent
stereoselectivity. In addition, we also explored the impact of replacing W116 in OYE 3
since the corresponding changes in OYE 1 greatly impacted stereoselectivity.
Project Summary
One goal of this project was to crystallize and solve the crystal structure of S.
cerevisiae OYE 3. We initially attempted to crystallize a GST-OYE 3 fusion protein. The
GST tag simplifies protein purification and allows for a similar high throughput isolation
strategy analogous to that of a His-tag.82 A GST-OYE 3 fusion protein would stream-line
the cloning and allow us to make constructs for both easy protein purification and
crystallization. In parallel, we also explored crystallization conditions for the native OYE
3 protein (lacking the GST-tag). We also explored mutagenesis of W116, the analogous
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position to W116 in OYE 1 and I113 in OYE 2.6 (the significance of these two positions
was mentioned extensively in Chapter 2). We created a single 96 well plate containing
the complete collection of OYE 3 W116 mutants and then screened these variants
against a set of substrates (Figure 4-6 through 4-8). Finally, we also attempted to
crystallize two OYE 1 double mutants related to the sequence of OYE 3 (OYE 1 W116A
/ F296S and W116V / F296S). Because neither OYE 1 double mutant yielded usable
crystals, we carried out the analogous study using two OYE 3 single mutants (W116A
and W116V).
Results and Discussion
Crystallization of OYE 3
We first explored the possibility of crystallizing S. cerevisiae OYE 3 as a GST-
fusion protein since this would greatly simplify protein isolation. We successfully purified
the target fusion protein by our standard methods (glutathione affinity chromatography
followed by gel filtration). Crystallization trials were set up using several commercial
screening kits; however, few conditions provided even quasi crystals of sufficient quality
to warrant further optimization. Based on these results, the best initial hit conditions
involved 5 mg/mL GST-OYE 3 in 100 mM Citrate, 20% (v/v) PEG 4000, 18%
isopropanol, pH 5.5 and grown at room temperature for 18 days. Unfortunately even
these conditions provided only quasi crystals which were not close to diffraction quality.
After these unsuccessful results, we turned our attention to crystallizing native OYE 3.
We subcloned the OYE 3 gene from the GST-tagged construct in plasmid pDJB6
by a process that involved several site-directed mutations to remove undesired
restriction enzyme sites. The resulting gene was introduced into plasmid pET22b for
native overexpression (plasmid pRP4). We carried out a small scale preliminary study to
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predict whether OYE 3 would bind to the phenol affinity column originally developed to
purify native OYE 1. We tested the ability of OYE 3 to reduce 5 mM 2-methyl-
cyclohexen-1-one (substrate 12) in the presence and absence of 5 mM 4-chlorophenol
(Figure 4-9). This phenol commonly inhibits OYE homologs; moreover this is the same
functional group found on our affinity column. Cells harboring pRP4 were grown until
they reached mid log phase then they were induced overnight using auto-induction
media.81 Cells were harvested and lysed by the addition of lysozyme. Lysates were
combined with KPi buffered reaction mixtures, which were run overnight. Reactions
were quenched with ethyl acetate and the organic phase was then extracted for
analysis by GC-MS. The reactions lacking 4-chlorophenol showed complete reduction
of the enone while those containing 4-chlorophenol showed no conversion. These
results implied that the phenol affinity column developed for OYE 1 would also be
successful for OYE 3.
Native OYE 3 was overexpressed in E. coli and purified by ammonium sulfate
fractionation followed by our standard affinity chromatography followed gel filtration
methods used for OYE 1. After screening approximately 300 crystallization conditions,
the only viable crystals were observed when low molecular weight PEG was the main
precipitant at near neutral pH values (pH 6). Further attempts to optimize around other
promising conditions were unsuccessful, producing either showers of aggregated
crystals or non-crystallized precipitant. The most promising initial conditions were
obtained using the PEGRx HT screening kit from Hampton research in wells B12, C6,
C9, and G3. All produced showers of microcrystals with spherical nucleation sites. Wells
B5, C5, C8, D5, and E6 all produced large crystals of better quality with fewer, but still
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multiple, nucleation sites. Conditions from wells A8 (100 mM MES monohydrate, 22%
v/v PEG 400, pH 6.0), A10 (100 mM sodium citrate tribasic dehydrate, 30% v/v
polyethylene glycol monomethyl ether 550, pH 5.0), and B6 (100 mM HEPES, 30%
PEG 1,000, pH 7.5) produced the highest quality of crystals with only single nucleation
sites. Crystals obtained from these conditions were rhombus shaped (Figure 4-10) and
had the gold color which is characteristic of both OYE 1 and OYE 2.6 crystals. We
attempted to optimize around the conditions in well A10 by arraying a matrix of
precipitants (15% v/v PEG 8,000, 18% v/v PEG 3,500, & 25% v/v PEG 400), pH values
(4.5, 5.0, 5.5, & 6.0) and different protein concentrations (20 and 10mg/mL). We also
attempted to optimize around the conditions in well B6 by arraying a matrix of
precipitants (30% v/v PEG 1,000, 24% v/v PEG 1,500, & 12% v/v PEG 3,000), pH
values (6.5, 7.0, 7.5, & 8.0) and protein concentrations (20 and 10 mg/mL). None of
these efforts yielded better-quality crystals. In fact, the further the conditions deviated
from the initial screening conditions, the worse the crystals became. We therefore chose
crystals for data collection from the A8 and A10 wells.
The most successful conditions were 100 mM MES monohydrate, 22% v/v PEG
400, pH 6.0 which provided crystals that diffracted to a maximum usable resolution of
1.8 Å using the 21-ID-G beamline at the Advanced Photon Source, Argonne National
Laboratory. The unit cell measured 61.214 107.762 141.071 Å and the crystals
belonged to space group P 21 21 21. The asymmetric unit contained two molecules and
a solvent content of 53.52% with a Matthews coefficient of 2.65 Å3/Dal.36
Data Reduction and Structure Solution
Reflection data for the OYE 3 structure without ligand were processed using
XDS83 to a resolution of 1.8 Å. Phases were obtained using the Phaser-MR utility of the
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PHENIX program suite38 by molecular replacement using a modification of S.
pastorianus OYE 1 (PDB code 1OYB) as the search model. All ligands and water
molecules were removed prior to molecular replacement. Inspection of the model
showed two OYE 3 chains present in the asymmetric unit. The best solution for the
space group was determined to be P 21 21 21 and the initial model was well defined
throughout the scaffold of the protein giving an Rfree value of 0.37. The C-terminal region
at both positions K398 and N399 was a notable exception and these two positions were
therefore removed from the initial search model. The initially calculated 2Fo-Fc and Fo-Fc
maps showed electron density patterns that could be easily identified as FMN. After one
round of simulated annealing refinement, the Rfree value dropped to 0.31. Further
refinement using the xyz coordinates, B-factors, real-space, and occupancies
refinement strategy features in PHENIX.refine as well as continued cycles of model
building using the structure validation tools in COOT produced a model with an Rfree of
0.22. Once the protein scaffold was established, the electron density in the OYE 3
active site was addressed. The FMN cofactor was modeled into the structure and
subsequent refinement provided an Rfree value of 0.19. Components from both the
crystallization conditions and the purification protocol were modelled into the active site
to identify any ligand present; however, none reasonably accounted for the electron
density observed by the 2Fo-Fc and Fo-Fc maps. Final refinement with chloride modeled
into the active site provided an Rfree value of 0.19.
Reflection data for OYE 3 with HPBA were processed using XDS83 to a
resolution of 1.9 Å. Phases were obtained using the Phaser-MR utility of the PHENIX
program suite38 by molecular replacement using a modification of S. pastorianus OYE 1
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(PDB code 1OYB) as the search model. All ligands and water molecules were removed
prior to molecular replacement. The best solution for the space group was determined
to be P 21 21 21 with an initial Rfree value of 0.38. Terminal positions K398 and N399
were truncated from model and one round of refinement using simulated annealing
followed reducing the Rfree value to 0.33. Further refinement using the xyz coordinates,
B-factors, real-space, and occupancies refinement strategy features in PHENIX.refine
as well as continued cycles of model building using the structure validation tools in
COOT reduced the Rfree to 0.25. The FMN cofactor was modeled into the structure and
subsequent refinement dropped the Rfree value to 0.24. Once the protein scaffold was
established, the electron density in the HPBA was modeled into the active site provided
and subsequent refinement reduced the Rfree value to 0.23. Further modeling lowered
the Rfree value to 0.22.
Reflection data for OYE 3 W116V were processed using XDS83 to a resolution of
1.9 Å. Phases were obtained using the Phaser-MR utility of the PHENIX program suite38
by molecular replacement using a modification of S. pastorianus OYE 1 (PDB code
1OYB) as the search model. All ligands and water molecules were removed prior to
molecular replacement. The best solution for the space group was determined to be P
21 21 21 with an initial Rfree value of 0.39. Terminal positions K398 and N399 were
truncated from model and one round of refinement using simulated annealing followed
reducing the Rfree value to 0.33. The FMN cofactor was modeled into the structure and
subsequent refinement reduced the Rfree value to 0.32. Further refinement using the xyz
coordinates, B-factors, real-space, and occupancies refinement strategy features in
PHENIX.refine as well as continued cycles of model building using the structure
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validation tools in COOT dropped the Rfree to 0.24. Once the protein scaffold was
established, the electron density in the OYE 3 active site was addressed. No HPBA
ligand was bound within the active site, instead the active site had water analogous to
that of the OYE 1 structure 1OYA and 1OYC. Final refinement with water modeled into
the active site provided an Rfree value of 0.24.
The overall structure of OYE 3 is nearly identical to that of OYE 1. OYE 3
structure has an analogous alpha beta barrel with the active site located within the
barrel (Figure 4-11). The FMN environment of OYE 3 was similar to that of OYE 1, with
amino acids T37, G72, Q114, and R243 hydrogen bonding to the FMN (Figure 4-1).
Residues H191 and N194 were positioned in a way that would allow them to form
hydrogen bonds to the carbonyl of a bound substrate, analogous to their roles in OYE 1
(Figure 4-2). Interestingly, nearly all active site residues were identical between OYE 1
and OYE 3, with the exception of position 296 (Ser in OYE 3 and Phe in OYE 1). A
complete list of the measured distances between β-carbons of each active site residue
to the closed carbon on the bound ligand are listed in Table 4-3. Position 296 is located
on loop 6, which opens during the catalytic cycle. Phenylalanine 296 in OYE 1 extends
into the active site when the loop is closed, taking up significantly more space than
S296 in OYE 3, which directs its side-chain outward, toward the external solvent (Figure
4-12). Efforts to understand how this sequence difference at position 296 impacts the
stereoselectivities of OYE 1 and OYE 3 are ongoing and involve molecular dynamics
simulations of both enzymes (a collaboration with Professor Adrian Roitberg and his
group).
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OYE 3 W116 Site Saturation Mutagenesis
After successfully solving the crystal structure of OYE 3, we created all possible
variants at position 116 using the non-GST-tagged OYE 3. The wild-type and all
mutants were screened against a series of substrates that had been investigated in
previous studies in order to obtain comparative data (Figure 4-15 through 4-28).22,24,58
In the case of cyclopentenone 1, OYE 3 W116H and W116Y showed the best
conversion and (S)-selectivitivty while OYE 3 W116E provided almost racemic product
(the greatest level of (R)-product observed from the mutant collection). Wild-type OYE 3
showed no significant conversion for 1.
In the case of cyclohexenone 2, OYE 3 W116H and W116Q yielded the most
(S)-product while OYE W116E afforded the highest level of (R)-product. As before wild-
type OYE 3 did not reduce cyclohexenone 2.
Neither wild-type nor any W116 mutant reduced pulegone 3. In the case of (S)-
and (R)-carvone (4 and 5),the wild-type and all W116 variants gave the same
stereochemical outcomes. This stands in marked contrast to the results with OYE 1, for
which changing W116 dramatically altered the stereochemical course of carvone
reductions.24 This points to an interplay between the residues at position 116 and 296,
despite the long distance between them (18.6 Å).
Neither wild-type OYE 3 nor any of the W116 mutants gave significant
conversion for substrates 11, 13, 14 or 15. Enones 21 and 22 were efficiently reduced
by OYE 3 wild-type and all W116 variants; however, highly hindered substrates 23 and
24 gave poor conversion with the same proteins. The best were OYE 3 W116P and
W116T for cyclohexenone 23 and OYE 3 W116C for spirocycle 24. While many of the
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OYE 3 W116 mutants gave conversion for substrate 25 (with the best provided by the
W116N variant), none yielded the opposite stereoisomer.
X-Ray Crystallography Studies of OYE 3 W116 Mutants and Related Proteins
Our collaborators asked us to solve the structures of two OYE 1 variants whose
sequences were related to that of OYE 3: OYE 1 W116A / F296 S and W116V / F296S.
Unfortunately, neither of these double mutants bound to the phenol affinity column
sufficiently strongly to allow purification. Instead, both eluted from the column prior to
dithionite addition. This behavior suggests significant changes to the active site
structure and / or the local environment of the FMN. The small fraction of protein that
did bind to the affinity column was collected and further purified by gel filtration. The
isolated proteins immediately precipitated from solution after mixing with the standard
OYE 1 crystallization buffer, yielding a brown, insoluble mass. His-tagged OYE 1
mutants were subsequently tested, but fared no better under our standard OYE 1
crystallization conditions. We also used an older purification protocol for OYE 1 based
on anion exchange chromatography.5 Unfortunately, the double mutants were not
sufficiently robust to survive this purification and a significant amount of the protein
precipitated prior to transfer to crystallization buffer. The remainder precipitated in the
crystallization buffer itself.
Interestingly, the single mutant OYE 1 F296S bound avidly to the phenol affinity
column and crystallized without incident. Moreover, our group has previously solved the
crystal structures of the single mutants OYE 1 W116A and OYE 1 W116V without
incident.24,84 We must therefore conclude that the combined effect of mutations at
positions 116 and 296 affect the active site so significantly in OYE 1 that they prevent
phenol binding and significantly diminish protein stability.
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Because OYE 3 can be considered (at least formally) analogous to the OYE 1
F296S single mutant, and we had previously prepared clones for OYE 3 W116A and
W116V, we used these variants as models for the OYE 1 double mutants. Both OYE 3
mutants could be purified by phenol affinity chromatography and crystallized under the
standard conditions developed for wild-type OYE 3. Crystals of OYE 3 W116V were
soaked with p-hydroxybenzaldehyde and sent for data collection. The OYE 3 W116V
crystal successfully diffracted and the structure was determined. Unfortunately the
soaking was unsuccessful and p-hydroxybenzaldehyde did not bind to the active site.
For comparison, we overlapped the OYE 3 structure with p-hydroxybenzaldehyde
bound to the OYE 3 W116V structure. Substitution of valine at position W116 decreased
the distance from the C2 of p-hydroxybenzaldehyde to the β-carbon of the residues by
0.1 Å (Figure 4-13). With a valine mutation however, the distance from the edge of the
residue (γ-carbon for valine and the ζ-carbon for tryptophan) to the C2 of p-
hydroxybenzaldehyde increased by 2.7 Å.
OYE 1 F296S was also crystalized and solved for comparison. Crystals of OYE 1
F296S were also soaked with p-hydroxybenzaldehyde and sent for data collection. The
crystal successfully diffracted and the structure was determined. Unfortunately the
soaking was unsuccessful for OYE 1 F296S as well and p-hydroxybenzaldehyde did not
bind to the active site. The serine at position F296S is orientated in the same manner as
position S296 in OYE 3. There appears to be no significant difference in the orientation
of loop 6 in OYE 1 F296S due to the substitution of a serine. For comparison, we
overlapped the OYE 1 structure with p-hydroxybenzaldehyde bound (1OYB) to the OYE
1 F296S structure and OYE 3. Substitution of serine at position F296 made no
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difference in the distance (7.8 Å) from the C4 of p-hydroxybenzaldehyde to the β-carbon
of the 296 residues (Figure 4-14). With a serine mutation however, the distance from
the edge of the residue (β-carbon for serine and the ζ-carbon for phenylalanine) to the
C4 of p-hydroxybenzaldehyde increased by 3.2 Å.
Experimental
General
Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained
from Integrated DNA Technologies. Crystallography screening kits (Classics Suite/
AmSO4 and PEGRx HT) were purchased from Qiagen and Hampton Research,
respectively. All other reagents were obtained from commercial suppliers and used as
received. Plasmids were purified on small scales by Wizard® minicolumns (Promega
Life Sciences) and on large scales using CsCl density gradient ultracentrifugation. DNA
sequencing was carried out by the University of Florida ICBR using capillary
fluorescence methods using standard protocols. LB medium contained 5 g/L Bacto-
Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl. ZY medium contained 5 g/L
Bacto-Yeast Extract and 10 g/L Bacto-Tryptone. 50x5052 contained 25% glycerol, 2.5%
glucose, and 10% a-lactose monohydrate. NPS x20 contained 66 g/L (NH4)2SO4, 136
g/L KH2PO4, and 142 g/L Na2HPO4.
Cloning
Construction of plasmid used for libraries
Plasmid construction began with pDJB6, a construct made by the combined
efforts of former group members Iwona Kaluzna and Despina Bougioukou. This plasmid
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was constructed from pIK2 (a pYEX-41 derivative containing a GST tag) with pET26a
and adding the wild-type S. cerevisiae OYE 3 gene.85 In this study, a silent mutation
was made at position Y389 in the OYE 3 gene to remove an internal NdeI site (primers
are listed as Y389Y Fwd and Rev in Appendix A-4).26,27 The PCR product was purified
by a Wizard® Plus SV Gel PCR Clean up kit (Promega), transformed into ElectroTen-
Blue® E. coli cells and the desired silent mutation was verified by Sanger sequencing.
The resulting plasmid was designated pRP1. Cloning continued with the removal of a
second internal NdeI site which preceded the GST gene using PCR. This yielded
plasmid pRP2. A unique NdeI site was introduced at the start of the OYE 3 coding
region using PCR, resulting in plasmid pRP3. Plasmid pRP3 was prepared on a large
scale by CsCl density gradient29 and the gene encoding the native OYE 3 protein was
excised by digesting with NdeI and XhoI. The fragment was inserted into pET22b,
previously cut with NdeI and XhoI, using T4 DNA ligase to yield pRP4 (Appendix E,
Figure E-4). Products from the ligation reaction were transformed into ETB cells and
sequenced to verify the desired sequence of pRP4. This was used to transform E. coli
overexpression strain BL21 (DE3) Gold using electro-transformation.
Construction of an OYE 3 W116 site-saturation mutagenesis library
Plasmid pRP4 was the template used to make W116 mutants in OYE 3. A set of
mutagenic primers containing a single codon replacement at position W116 was used to
make each of the 19 mutants (primers are listed in Appendix A, Table A-4). PCR was
performed using 0.5 μL of template (18 ng/µL), 5 μL of both forward and reverse
mutagenic primers (5 mM), 1 μL of dNTP mix (10 mM), 10 μL of 5X HF Phusion® Hot
start buffer, 28 μL of sterile water, and 0.5 μL of Phusion® Hot Start II DNA Polymerase
(2 U/μL) for a total reaction volume of 50 μL. PCR was performed using a MJ Mini®
148
thermocycler from BioRad and samples were run with an initial denaturation step at
98°C for 30 s, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 64°C
for 30 s, and an extension step at 72°C for 3 min 30 s, after which the reactions were
completed with a final extension step at 72°C for 7 min 30 s.
All PCR samples were purified by Wizard® Plus SV Gel PCR Clean up kits
(Promega), using the manufacturer instructions. Samples were then incubated overnight
with two 0.5 μL aliquots of 20 U/μL DpnI at 37°C to remove the parent template. The
first aliquot was added immediately after PCR clean up and the second was added after
4 h of digestion. After DpnI digestion, samples were purified using Wizard® Plus SV Gel
PCR Clean up kits.
DNA samples were used to transform E. coli ETB cells using a Gene Pulser®
from BioRad. Electroporation was carried out with 4 μL of DNA sample and 50 µL of
ETB cells with a voltage of 2.5 kV. Cells were then incubated in 600 μL of SOC medium
at 37°C for 1 h before plating onto LB-amp agar plates and growing at 37°C for 36 h.
Plasmids from randomly-chosen colonies were sequenced to verify that the desired
mutation was present using Sanger sequencing. The desired plasmids were then used
to transform E. coli expression strain BL 21 (DE3) Gold. This was accomplished using
electroporation (2.5 kV) with 4 μL of plasmid (10 ng/μL) and 80 μL of E. coli BL21 (DE3)
Gold electrocompetent cells. Electroporated samples were incubated in 600 μL of SOC
medium at 37°C for 45 min prior to plating onto LB-amp agar plates and growing
overnight at 37°C. Mutations were verified by sequencing, then transformants were
assembled into a 96 well microtiter plate. Transformants were grown in 600 μL of LB-
amp in a 96 well microtiter plate overnight to reach saturation. The library was
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completed with the transfer of 120 μL of saturated cultures into a new 96 well microtiter
plate containing 30 μL of 80% glycerol which brought the final concentration of glycerol
to 15%.
Testing of Phenol Binding to OYE 3
E. coli harboring pRP4 were taken from cell streaks on an LB-amp agar plate and
grown in 5 mL of LB-amp overnight to reach saturation. A 500 μL aliquot of this
preculture was used to inoculate a solution containing 50 mL of ZYP-5052 auto-
induction media and grown overnight. Cells were harvested and resuspended in 5 mL of
50 mM KPi, pH7 containing 1 mg/mL lysozyme. Cells were incubated in the lysozyme
solution for 1 h at 37°C. Samples were then centrifuged at 12,000 × g to remove the
insoluble debris. An aliquot of 200 μL of lysate was added to a pair of 500 μL reaction
mixtures containing 50 mM KPi, pH 7, 5 mM substrate 12, 100 mM glucose, 44 U GDH,
0.3 mM NADP+. One of the two reaction mixtures was supplemented with 5 mM 4-
chlorophenol. Reactions were run overnight at 30°C before quenching with 0.5 mL of
ethyl acetate. The organic phase was extracted and analyzed by GC-MS by method
JON.Meth (Appendix D) using a DB-17 column.
GST-OYE 3 Fusion Protein Purification and Crystallogenesis
E. coli BL21 (DE3) harboring plasmid pDJB6 (a derivative of pET22b with an
GST-OYE 3 fusion coding region flanked by NdeI and XhoI sites) was grown at 37°C in
a 4 L fermenter containing LB medium supplemented with 36 µg/mL kanamycin (LB-
kan). Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid
log phase then induced by adding IPTG to a final concentration of 0.4 mM and grown
with 100 mM glucose at 30°C with 600 rpm of agitation for 3 h. The cell solution was
then chilled at 4°C for 30 min before centrifugation at 5,000 × g. The wet cell pellet was
150
then resuspended in 1 mL of 1× PBS buffer per gram of wet cell pellet. Cells were then
lysed under 12,000 psi with the aid of a French Press. The lysate was centrifuged for 1
h at 15,000 × g. Portions of 10 mL of the supernatant were loaded onto a glutathione
column charged with 1× PBS buffer. Samples were eluted with 20 mL of an elution
buffer containing 50 mM Tris with 3 mg/mL of reduced glutathione at pH 7.5. The eluant
was further purified by gel filtration with a Superdex 200 column (Pharmacia) using 50
mM Tris-Cl, 50 mM NaCl, pH 7.5. Pooled elutant samples were then concentrated by
ultrafiltration to a final concentration of 20 mg/mL using Amicon tubes. Protein
concentration was determined by absorbance at 280 nm using an extinction coefficient
(ε) and molecular weights (MW) estimated by protparam (GST-OYE 3 fusion protein
had an ε of 106,480 M-1cm-1 with a MW of 68,543 Da).35
Protein crystals were screened using the PEGRx HT screening kit from Hampton
research as well as the Classics suite, and AmS04 suite kits from Qiagen. Wells
contained 2 μL of crystallization solution combined with 2 μL of 20 mg/mL GST-OYE 3
fusion protein in 50 mM Tris-Cl, 50 mM NaCl buffer pH 7.5 and used sitting drop vapor
diffusion. The best crystals obtained were quasi crystals obtained from well G4 of
PEGRx HT screening kit. These crystals were grown for 18 days with 5 mg/mL GST-
OYE 3 in 0.1 M Citrate, 20% (w/v) PEG 4000, 18% isopropanol, at pH 5.5 at room
temperature.
Native OYE 3 Protein Purification and Crystallogenesis
E. coli BL21 (DE3) Gold harboring plasmid pRP4 (a derivative of pET22b with an
OYE 3 coding region flanked by NdeI and XhoI sites) was grown at 37°C in a 4 L
fermenter containing LB medium supplemented with 200 μg/mL ampicillin (LB-amp).
Cells were grown in the fermenter with 600 rpm of agitation for 2 h to achieve mid log
151
phase, then induced by adding IPTG to a final concentration of 0.4 mM and grown with
100 mM glucose at 30°C with 600 rpm of agitation for 3 h. The cell solution was then
chilled at 4°C for 30 min before centrifugation at 5,000 × g. The wet cell pellet was then
resuspended in 1 mL of 100 mM Tris-Cl, pH 8.0 containing 10 μM PMSF per gram of
wet cell pellet. Cells were then lysed under 12,000 psi with the aid of a French Press.
The lysate was centrifuged for 1 h at 15,000 × g. Nucleotides were precipitated out of
supernatant by adding protamine sulfate to a final concentration of 1 mg/mL and stirring
for 20 min at 4°C. The supernatant was separated by centrifugation at 15,000 × g for 20
min. Proteins were precipitated by adding 5 portions of solid ammonium sulfate every 5
min to achieve a concentration of 78% saturation. The pellet was recovered by
centrifuging at 15,000 × g for 1 hr.
The ammonium sulfate pellet was resuspended in 100 mM Tris-Cl, 100 mM
(NH4)2SO4, 10 μM PMSF, pH 8.0, then dialyzed against 1 L of this buffer overnight at
4ºC. The following day the sample was then dialyzed for 2 h against 1 L of the same
buffer containing 10 mM sodium dithionite. After 2 h the buffer was exchanged for a
fresh 1 L of buffer containing 10 mM sodium dithionite and dialysis continued for 2 h.
The sample was then transferred to a fresh 1 L of buffer without dithionite and dialyzed
for 2 h, after which, the buffer was exchanged with a final 1 L of fresh buffer and
dialyzed overnight. The final sample was then centrifuged at 15,000 g for 30 min to
remove any insoluble debris accumulated during dialysis, then it was applied to a N-(4-
hydroxybenzoyl) aminohexyl agarose affinity column with the aid of an FPLC.
The affinity column was packed with 3 mL of the matrix and was equilibrated with
100 mM Tris-Cl, 100 mM (NH4)2SO4, 10 μM PMSF buffer at pH 8.0, prior to use. The
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flow rate during purification was 0.5 mL/min. Elution of non-bound protein was
monitored by absorbance at 280 nm and a baseline established prior to loading protein
sample onto the column. Dialyzed protein samples were loaded onto the affinity column
in 10 mL portions. OYE 3 binding to the column gave a green color with a slightly bluer
tint than observed for OYE 1. Bound OYE 3 was washed with starting buffer until 280
nm absorbance returned to the baseline, this required anywhere between 30-60 mL of
buffer. The OYE 3 was eluted by washing with 100 mM Tris-Cl, 100 mM (NH4)2SO4, 10
μM PMSF, 4 mM sodium dithionite, pH 8.0. The collected protein sample was further
purified by gel filtration with a Superdex 200 column (Pharmacia) using 50 mM Tris-Cl,
50 mM NaCl, pH 7.5 at a flow rate of 0.5 mL/min. Fractions containing OYE 3 were
combined and concentrated by ultrafiltration to a final concentration of 40 mg/mL using
an Amicon tube. Protein concentration was determined by absorbance at 280 nm using
an extinction coefficient (ε) and molecular weights (MW) estimated by protparam (OYE
3 had an ε of 76,905 M-1cm-1 with a MW of 44,920 Da).
Crystallization conditions were screened using the PEGRx HT screening kit from
Hampton research as well as the Classics and AmS04 suite kits from Qiagen. Wells
contained 2 μL of crystallization solution combined with 2 μL of 20 mg/mL OYE 3 in 50
mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop vapor diffusion. The best crystals
were obtained after 14 days using 100 mM MES monohydrate, 22% (v/v) PEG 400, pH
6.0 (well A8 of the PEG RX screening kit) at room temperature. No further optimization
of these crystallization conditions was required. Crystals were soaked in a harvesting
buffer for 5 s (100 mM MES monohydrate, 22% v/v PEG 400, 15% (v/v) glycerol, pH
6.0) before being flash cooled in liquid nitrogen and sent for synchrotron data collection.
153
OYE 3 with bound p-HBA was crystallized using the PEGRx HT screening kit
from Hampton research. Wells contained 2 μL of crystallization solution combined with 2
μL of 16 mg/mL OYE 3 in 50 mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop
vapor diffusion. The best crystals were obtained after 10 days using 100 mM sodium
citrate tribasic dihydrate, 30% (v/v) polyethylene glycol monomethyl ether 550, pH 5.0
(well A10 of the PEG RX screening kit) at room temperature. The ligand soaking
occurred during harvesting. Crystals were soaked in harvesting buffer containing 2 mM
p-HBA for 5 s (100 mM Sodium citrate tribasic dihydrate, 30% (v/v) polyethylene glycol
monomethyl ether 550, 2 mM p-HBA, 15% (v/v) glycerol, pH 5.0) before being flash
cooled in liquid nitrogen and sent to the synchrotron for data collection.
OYE 3 W116V was crystallized using a PEGRx HT screening kit from Hampton
research. Wells contained 2 μL of crystallization solution combined with 2 μL of 27
mg/mL OYE 3 in 50 mM Tris-Cl, 50 mM NaCl, pH 7.5 and used sitting drop vapor
diffusion. The best crystals were obtained after 30 days using 100 mM sodium citrate
tribasic dihydrate, 30% (v/v) polyethylene glycol monomethyl ether 550, pH 5.0 (well
A10 of the PEG RX screening kit) at room temperature. The ligand soaking occurred
during harvesting. Crystals were soaked in harvesting buffer containing 2 mM p-HBA for
5 s (100 mM Sodium citrate tribasic dihydrate, 30% (v/v) polyethylene glycol
monomethyl ether 550, 2 mM p-HBA, 15% (v/v) glycerol, pH 5.0) before being flash
cooled in liquid nitrogen and sent for synchrotron data collection. Ligand soaking was
unsuccessful.
OYE 1 F296S was crystallized using the published conditions.20 Wells contained
6 μL of crystallization solution combined with 6 μL of 20 mg/mL OYE 1 F296 in 50 mM
154
Tris-Cl, 50 mM NaCl, pH 7.5 and used hanging drop vapor diffusion. The best crystals
were obtained after 7 days using 35% (v/v) PEG 400, 100 mM Na HEPES, 200 mM
MgCl2 buffer pH 8.3 at 6°C. The ligand soaking occurred during harvesting. Crystals
were soaked in harvesting buffer containing 2 mM p-HBA for 5 s (35% (v/v) PEG 400,
100 mM Na HEPES, 200 mM MgCl2, 2 mM p-HBA, 15% (v/v) glycerol, pH 8.3) before
being flash cooled in liquid nitrogen and sent for synchrotron data collection. Ligand
soaking was unsuccessful.
Alkene Substrates for OYE 3
A list of substrates and products is shown if Figure 4-6 through 4-8.
2-(Hydroxymethyl)-cyclopent-2-enone (1)
2-(Hydroxymethyl)-cyclopent-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Kar and Argade.30 2-(Hydroxymethyl)-
cyclopent-2-enone can be detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm x 30 m). The temperature program began with an initial temperature
of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at
which the program remained for 5 min (GC method is listed as AZW2.Meth in Appendix
D). 2-(Hydroxymethyl)-cyclopent-2-enone eluted near 13.1 min. The reduced products
(S)- and (R)-6 eluted near 11.4 and 10.2 min, respectively.
2-(Hydroxymethyl)-cyclohex-2-enone (2)
2-(Hydroxymethyl)-cyclohex-2-enone was prepared in our lab by Bradford
Sullivan28 using the method developed by Rezgui and El Gaied.31 2-(Hydroxymethyl)-
cyclohex-2-enone was detected during screening by GC-FID using a Beta Dex 225
column (0.25 mm x 30 m). The temperature program began with an initial temperature
of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at
155
which the program remained for 5 min (GC method is listed as AZW2.Meth in Appendix
D). 2-(Hydroxymethyl)-cyclohex-2-enone eluted near 13.1 min. The reduced products
(S)- and (R)-7 eluted near 10.2 and 10.8 min, respectively.
(R)-Pulegone (3)
(R)-Pulegone was purchased from Sigma Aldrich and can be detected during
screening by GC-MS using a DB-17 column (0.25 mm x 30 m). The temperature
program began with an initial temperature of 90°C, followed by an increase at 10°C/min
to a temperature of 130°C, followed by an increase at 2°C/min to a temperature of
150°C, followed by an increase at a rate of 20°C/min to a temperature of 250°C, at
which the program remained for 5 min (GC method is listed as YAP.Meth in Appendix
D). (R)-Pulegone eluted near 8.44 min and the reduced products cis- and trans-8 elute
at 6.01 min and 6.37 min (unassigned).
S-(+)-Carvone (4)
S-(+)-Carvone was purchased from Sigma Aldrich and it can be detected by GC-
MS using a DB-17 column (0.25 mm x 30 m). The temperature program began with an
initial temperature of 90°C, followed by an increase at 10°C/min to a temperature of
130°C, followed by an increase at 2°C/min to a temperature of 150°C, followed by an
increase at a rate of 20°C/min to a temperature of 250°C, at which the program
remained for 5 minutes (GC method is listed as YAP.Meth in Appendix D). S-(+)-
Carvone eluted near 8.8 min. A mixture of reduced product isomers, (+)-Dihydrocarvone
(Acros) was used as a standard to assign the peaks for both cis- and trans-9 (7.6 and
7.2 min, respectively).
156
R-(-)-Carvone (5)
R-(-)-Carvone was from Sigma Aldrich and can be detected by GC-MS using a
DB-17 column (0.25 mm x 30 m). The temperature program began with an initial
temperature of 90°C, followed by an increase at 10°C/min to a temperature of 130°C,
followed by an increase at 2°C/min to a temperature of 150°C, followed by an increase
at a rate of 20°C/min to a temperature of 250°C, at which the program remained for 5
min (GC method is listed as YAP.Meth in Appendix D). R-(-)-Carvone eluted near 8.8
min. A mixture reduced product isomers, (+)-Dihydrocarvone (Acros) was used as a
standard to assign the peaks for both cis- and trans-10 (7.6 and 7.2 min, respectively).
2-Methyl-2-cyclopenten-1-one (11)
2-Methyl-2-cyclopenten-1-one was from Sigma Aldrich and can be detected
during by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature
program began with an initial temperature of 100°C for 10 min, followed by an increase
at 20°C/min to a temperature of 180°C, at which the program remained for 3 min. (GC
method is listed as BTS2.Meth in Appendix D). 2-Methyl-2-cyclopenten-1-one eluted
near 9.1 min and the reduced products (S)- and (R)-16 eluted near 5.9 min and 5.7 min
(unassigned).
2-Methyl-2-cyclohexen-1-one (12)
2-Methyl-2-cyclohexen-1-one was from Sigma Aldrich and was detected by GC-
MS using a DB-17 column (0.25 mm x 30 m). The temperature program began with an
initial temperature of 60°C for 2 min, followed by an increase at 10°C/min to a
temperature of 195°C, at which the program remained for 10 min (GC method is listed
as JON.Meth in Appendix D). 2-Methyl-2-cyclohexen-1-one eluted near 8.5 min and the
reduced product 2-methylcyclohexanone (Sigma Aldrich) eluted near 7.2 min.
157
3-Methyl-cyclohexen-1-one (13)
3-Methyl-cyclohexen-1-one was commercially available and can be detected by
GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature program
began with an initial temperature of 70°C for 2 min, followed by an increase at
0.3°C/min to a temperature of 90°C which then immediately increased at 20°C/min to
temperature of 180°C, at which the program remained for 3 min. (GC method is listed
as BTS4.Meth in Appendix D). 3-Methyl-cyclohexen-1-one eluted near 73.1 min and the
reduction products (S)- and (R)-18 eluted near 47.4 and 48.2 min, respectively.
3-Ethyl-cyclohexen-1-one (14)
3-Ethyl-cyclohexen-1-one was prepared by in our lab by Magdalena Swiderska
using the method developed by Chandrasekhar and Reddy.12,86 3-Ethyl-cyclohexen-1-
one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The
temperature program began with an initial temperature of 70°C for 2 min, followed by an
increase at 0.3°C/min to a temperature of 90°C which then immediately increased at
20°C/min to temperature of 180°C, at which the program remained for 3 min. (GC
method is listed as BTS4.Meth in Appendix D). 3-Ethyl-cyclohexen-1-one eluted near
73.5 min and the reduction products (S)- and (R)-19 eluted near 59.7 and 62.2 min,
respectively.
3-Methyl-cyclopenten-1-one (15)
3-Methyl-cyclopenten-1-one was from Sigma Aldrich and can be detected during
screening by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature
program began with an initial temperature of 90°C for 20 min, followed by an increase at
20°C/min to a temperature of 180°C, at which the program remained for 3 min. (GC
method is listed as BTS3.Meth in Appendix D). 3-Methyl-cyclopenten-1-one eluted near
158
24.9 min and the reduction products (S)- and (R)-20 eluted near 15.2 and 15.7 min,
respectively.
4-Ethyl-4-methyl-2-cyclohexen-1-one (21)
4-Ethyl-4-methyl-2-cyclohexen-1-one was prepared in our lab by Bradford
Sullivan using the method developed by Flaugh et al.58 4-Ethyl-4-methyl-2-cyclohexen-
1-one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The
temperature program began with an initial temperature of 100°C for 30 min, followed by
an increase at 20°C/min to a temperature of 180°C, at which the program remained for
3 min. (GC method is listed as BTS7.Meth in Appendix D). 4-Ethyl-4-methyl-2-
cyclohexen-1-one eluted near 29.5 min and the reduced product 26 eluted near 21.4
min.
4-Isopropyl-4-methyl-2-cyclohexen-1-one (22)
4-Isopropyl-4-methyl-2-cyclohexen-1-one was prepared in our lab by Bradford
Sullivan using the method developed by Flaugh et al.58 4-Isopropyl-4-methyl-2-
cyclohexen-1-one can be detected by GC-FID using a Beta Dex 225 column (0.25 mm x
30 m). The temperature program began with an initial temperature of 100°C for 30 min,
followed by an increase at 20°C/min to a temperature of 180°C, at which the program
remained for 3 min. (GC method is listed as BTS7.Meth in Appendix D). 4-Isopropyl-4-
methyl-2-cyclohexen-1-one eluted near 33.4 min and the reduced product 27 eluted
near 31.6 min.
4,4-Diethyl-2-cyclohexen-1-one (23)
4,4-Diethyl-2-cyclohexen-1-one was prepared in our lab by Bradford Sullivan
using the method developed by Flaugh et al.58 4,4-Diethyl-2-cyclohexen-1-one can be
detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature
159
program began with an initial temperature of 100°C for 15 min, followed by an increase
at 20°C/min to a temperature of 195°C, at which the program remained for 10 min. (GC
method is listed as BTS8.Meth in Appendix D). 4,4-Diethyl-2-cyclohexen-1-one eluted
near 20.1 min and the reduced product 28 eluted near 19.7 min.
Spiro[5.5]undec-1-en-3-one (24)
Spiro[5.5]undec-1-en-3-one was prepared in our lab by Bradford Sullivan using
the method developed in by Flaugh et al.58 Spiro[5.5]undec-1-en-3-one can be detected
by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature program
began with an initial temperature of 100°C for 15 min, followed by an increase at
20°C/min to a temperature of 195°C, at which the program remained for 10 min. (GC
method is listed as BTS8.Meth in Appendix D). Spiro[5.5]undec-1-en-3-one eluted near
23.4 min and the reduced product 29 eluted near 22.7 min.
2-Butylidenecyclohexanone (25)
2-Butylidenecyclohexanone was prepared in our lab by Magdalena Swiderska
using a method developed by Huang et al.12,87 2-Butylidenecyclohexanone can be
detected by GC-FID using a Beta Dex 225 column (0.25 mm x 30 m). The temperature
program began with an initial temperature of 70°C with an increase at 0.3°C/min to a
temperature of 100°C which then immediately increased at 20°C/min to temperature of
190°C, at which the program remained for 3 min. (GC method is listed as BTS10.Meth
in Appendix D). 2-Butylidenecyclohexanone eluted near 93.0 min and (S)- and (R)-30
eluted near 47.4 and 48.2 min, respectively.
Screening
E. coli BL21 (DE3) Gold cells harboring plasmids encoding wild-type and W116
site-saturation OYE 3 mutants were grown in a 96 well plate containing 600 μL of LB-
160
amp. Cells were grown at 37°C with 250 rpm of agitation overnight. The saturated
cultures were then used to inoculate a larger 2 mL square bottom 96 well plate. This
larger square bottom plate contained 600 μL of an auto-induction medium. The auto-
induction medium contained a mix of ZY media, 50x5052, 20x NPS, and 200 μg/mL
ampicillin.33 Cells were induced in an aeration case developed at the University of
Florida by the machine shop in the Department of Chemistry. Induction occurred at
37°C with 350 rpm of agitation overnight. The increased agitation is required for
induction to occur. Induced cells were then separated from the auto-induction medium
by centrifugation. Auto-induction medium was removed and induced pelleted cells were
then resuspended in 600 μL of a reaction mixture. The reaction mixtures contained 50
mM KPi buffer with 100 mM glucose and 15 mM of the substrate of interest at pH 7.0.
Reactions were run at room temperature with 250 rpm of agitation overnight. Reactions
were quenched by adding 500 μL of ethyl acetate. The organic phase was separated by
centrifugation and extracted for analysis by GC.
Conclusions
We discovered multiple crystallization conditions for wild-type OYE 3 and OYE 3
W116 mutants and we also solved the structure of OYE 3. Given the sequence identity
between OYE 3 and OYE 1 (80%), it is not surprising that these homologs also have
very similar structures. The only significant difference in the active site is the presence
of a serine at position 296 on loop 6 in OYE 3 (OYE 1 has a phenylalanine at position
296). Our results have highlighted the importance of this active site residue, which will
be the subject of future studies. There is significant potential for protein engineering of
both OYE 1 and OYE 3 within loop 6. Whether variants with a combination of mutations
at W116 along with loop 6 mutations at position 296 can provide synergy to open up
161
OYE homologs to new substrates is yet to be seen; however, we now have a complete
set of OYE 3 W116 site-saturation mutants to survey and the means to investigate their
structures.
With regard to the substrate profiling of OYE 3 and site-saturation mutants for
W116, we learned that OYE 3 retains its preference for binding both carvone
enantiomers in the “normal” substrate binding mode, regardless of the nature of the
residue at position 116. This stands in stark contrast to the case for the analogous OYE
1 W116 mutants, many of which gave “flipped” substrate binding. For example, OYE 1
the A, C, E, G, I, M, N, Q, S, T and V replacements for W116 all afforded trans-9, the
result of “flipped” substrate binding.24 The same mutations in OYE 3 mutants gave no
trans-9, and only the “normal”, cis-9 product was observed. For (R)-carvone, both the
OYE 1 W116A and W116V variants give the product of flipped binding (cis-10). The
corresponding mutants in OYE 3 provide a 1 : 1 mixture of diastereomers. One other
difference in stereopreference was observed for substrates 1 and 2. The W116E mutant
of OYE 1 yielded (S)-6 and (S)-7 with ca. 90% ee. The W116E mutant of OYE 3 gave
racemic mixtures of 6 and 7. Finding such variations between these very similar OYE
homologs is fortunate and suggests that novel variants with even more different
stereoselectivities may be accessible after further study of OYE 3.
162
Table 4-1. Crystallographic data collection and refinement statistics.
Structure title OYE 3 Wt OYE 3 W116V OYE 3 soaked in p-HBA
Ligand Soaked none none p-HBA
X-Ray Source 21-ID-G beamline 21-ID-G beamline 21-ID-G beamline
APS Argonne National
Laboratory
APS Argonne
National Laboratory
APS Argonne National
Laboratory
Space Group P 21 21 21 P 21 21 21 P 21 21 21
Unit Cell Dimensions
61.214 107.762 141.071
90 90 90
61.63 106.935 140.46
90 90 90
61.63 106.421 141.01 90
90 90
a, b, c (Å)
Resolution (Å) 38.88 - 1.798
(1.862 - 1.798)
24.98 - 1.88
(1.947 - 1.88)
24.92 - 1.88
(1.947 - 1.88)
Unique Reflections 87271 (8450) 76198 (7496) 76002 (7479)
Completeness (%) 1.00 (0.98) 0.99 (1.00) 1.00 (1.00)
Multiplicity 14.5 (14.3) 14.3 (14.8) 14.314.6 (14.7)
Rsym 0.09911 (0.6901) 0.1248 (0.5961) 0.122 (0.5929)
I/σ (I) 20.18 (3.95) 15.01 (4.25) 18.53 (5.34)
Rwork 0.1581 (0.2360) 0.1999 (0.2362) 0.1820 (0.2073)
Rfree 0.1916 (0.2684) 0.2392 (0.2956) 0.2185 (0.2536)
Ramachandran
Statistics
Favored (%) 97 97 97
Allowed (%) 2.7 2.9 3.2
Outliers (%) 0 0 0
Number of Protein,
Solvent, and Ligand
Atoms
6309, 795, 64 6261, 790, 62 6325, 792, 126
Average B Factors
(Å2) 20.78 22.52 18.53
Protein 20.14 22.34 18.16
Ligands 18.65 17.14 25.12
Solvent 26.96 26.42 22.35
Values in parentheses denote data for the highest resolution bin (1.862 - 1.798 Å, 1.947 - 1.88 Å, and 1.947 - 1.88 Å)
163
Table 4-2. Crystallographic data collection and refinement statistics.
Structure title OYE 1 F296S
Ligand Soaked none
X-Ray Source 21-ID-G beamline
APS Argonne National
Laboratory
Space Group P 43 21 2
Unit Cell Dimensions
141.5 141.5 42.54
90 90 90
a, b, c (Å)
Resolution (Å) 24.98 - 1.88
(1.92 - 1.85)
Unique Reflections 37426 (3651)
Completeness (%) 1.00 (0.99)
Multiplicity 3.8 (3.8)
Rsym 0.0587 (0.669)
I/σ (I) 19 (2.6)
Rwork 0.1843 (0.2756)
Rfree 0.2225 (0.3390)
Ramachandran
Statistics
Favored (%) 96
Allowed (%) 3.9
Outliers (%) 1.8
Number of Protein,
Solvent, and Ligand
Atoms
3172, 410, 32
Average B Factors
(Å2) 26.52
Protein 25.25
Ligands 19.58
Solvent 36.86
Values in parentheses denote data for the highest resolution bin (1.92 -1.85 Å).
164
Table 4-3. Distances between the β-carbon of each active site residue to the ligand.
OYE 1 Residue
β-carbon Distance (Å)
Ref. Carbon # OYE 3 Residue
β-carbon Distance (Å)
N194 4.1 1 N194 4.2
Y196 6.7 1 Y196 6.8
G72* 6.4 2 G72 6.3
W116 7.0 2 W116 7.6
L118 8.9 2 L118 9.2
H191 7.3, (7.4) 2, (1)** H191 7.7, (7.5)**
T37 4.0 3 T37 3.8
M39 9.4 3 M39 9.4
Y82 9.0 3 Y82 9.2
F374 9.1 4 F374 8.9
Y375 9.1 4 Y375 9.3
N294 9.4 5 D294 8.9
P295 5.1 5 P295 4.8
F296 7.8 5 S296 7.4
F249 6.8 6 F250 6.4
N250 8.3 6 N251 7.9
Distances are measured in angstroms from the β-carbon of the indicated residue to the nearest ring carbon of the bound ligand. Phenolic carbon is designated as 1 and proceeds clockwise as viewed from above the bound FMN cofactor.28 *Measurement taken from α-carbon **Position H191 in OYE 3 is closer to C1 than the C2, which is reverse in OYE 1.
165
Figure 4-1. Schematic illustration of the FMN environment in the active site of OYE homologs. The FMN (Gold) environment of OYE 1 (green), OYE 2.6 (violet), and OYE 3 (magenta). OYE 3 uses the same hydrogen bonding partners as OYE 1 to lock the FMN into place within the active site (T37, G72, Q114, and R243). OYE 3 also uses the same hydrophobic partners beneath the FMN and shown with blue circles (P35, L36, and I351) to lock it into position.
Figure 4-2. The mechanism of OYE 3. Mechanism for the reduction of a bound α-unsaturated carbonyl substrate (black) by a reduced FMN (gold) in the active site of OYE 3 (magenta). OYE 3 uses the same hydrogen bonding partners as OYE 1 (H191 and N194) to lock the carbonyl into position.
166
Figure 4-3. Diagram of the positions in the active site of OYE homologs. The active site positions of OYE 1 (green), OYE 2.6 (violet), and OYE 3 (magenta). All positions except S296 in OYE 3 have the same residues as in OYE 1.
167
Figure 4-4. Loop 6 in OYE homologs. This figure shows loop 6 in OYE 1 (green) and OYE 3 (magenta) along with FMN (yellow) and a bound p-HBA ligand. This portion of the active site has the most variability across the structures of OYE homologs. The loop 6 of OYE 3 allows for the largest amount of unoccupied space south of the bound substrate.
168
Hall et al. (2008)42
Stueckler et al. (May 2010)14
Figure 4-5. List of OYE 3 substrates and reported conversion from the literature.
169
Stueckler et al. (October 2010)44
Stueckler et al. (2011)88
Figure 4-5. (Continued).
170
Brenna et al. (June 2011)45
Brenna et al. (July 2011)46
Figure 4-5. (Continued).
171
Brenna et al. (December 2011)47
Tasnadi et al. (March 2012)51
Figure 4-5. (Continued).
172
Tasnadi et al. (June 2012)50
Durchschein et al. (2012)49
Figure 4-5. (Continued).
173
Brenna et al. (January 2012)48
Brenna et al. (January 2012)89
Brenna et al. (February 2012)90
Figure 4-5. (Continued).
174
Brenna et al. (2013)52
Knaus et al. (2014)91
Figure 4-5. (Continued).
175
Brenna et al. (March 2014)53
Brenna et al. (July 2014)54
Figure 4-5. (Continued).
176
Turrini et al. (2015)55
Figure 4-5. (Continued).
177
Figure 4-6. First set of substrates and theoretical binding mode products.
178
Figure 4-7. Second set of substrates and theoretical binding mode products.
179
Figure 4-8. Third set of substrates and theoretical binding mode products.
180
Figure 4-9. The reactions used to test phenol binding by OYE 3. Substrate 12 was used with and without 4-chlorophenol.
Figure 4-10. Crystals and crystallization conditions for of OYE 1 (left), OYE 2.6 (middle), and OYE 3 (right).
181
Figure 4-11. The structure of OYE 3. The OYE 3 structure has an alpha beta barrel with the FMN (Yellow) located within the barrel.
Figure 4-12. The active site for both OYE 1 (green) and OYE 3 (magenta) with bound FMN (yellow) and substrate (OYE 3 blue and OYE 1 green).
182
Figure 4-13. The active site for both OYE 3 (magenta) and OYE 3 W116V (orange) with bound FMN and p-HBA (cyan).
Figure 4-14. The active site for both OYE 1 (green) and OYE 1 F296S (orange) with bound FMN and p-HBA (cyan).
183
Figure 4-15. Results from screening the OYE 3 W116 site-saturation library against substrate 1.
Figure 4-16. Results from screening the OYE 3 W116 site-saturation library against substrate 2.
184
Figure 4-17. Results from screening the OYE 3 W116 site-saturation library against substrate 3.
Figure 4-18. Results from screening the OYE 3 W116 site-saturation library against substrate 4.
185
Figure 4-19. Results from screening the OYE 3 W116 site-saturation library against substrate 5.
Figure 4-20. Results from screening the OYE 3 W116 site-saturation library against substrate 11.
186
Figure 4-21. Results from screening the OYE 3 W116 site-saturation library against substrate 13.
Figure 4-22. Results from screening the OYE 3 W116 site-saturation library against substrate 14.
187
Figure 4-23. Results from screening the OYE 3 W116 site-saturation library against substrate 15.
Figure 4-24. Results from screening the OYE 3 W116 site-saturation library against substrate 21.
188
Figure 4-25. Results from screening the OYE 3 W116 site-saturation library against substrate 22.
Figure 4-26. Results from screening the OYE 3 W116 site-saturation library against substrate 23.
189
Figure 4-27. Results from screening the OYE 3 W116 site-saturation library against substrate 24.
Figure 4-28. Results from screening the OYE 3 W116 site-saturation library against substrate 25.
190
CHAPTER 5 IMPROVING THE SUBSTRATE RANGE OF AMINOLEVULINIC ACID SYNTHASE
THROUGH PROTEIN ENGINEERING
Background
Enzymes that utilize pyridoxal phosphate (PLP) cofactors can perform an
impressive set of reactions. 5-Aminolevulinic acid synthase (ALAS, EC 2.3.1.37) is a
PLP dependent enzyme that performs a Claisen-like condensation between succinyl-
CoA and glycine to produce 5-aminolevulinic acid (δ-AL, ALA) (Figure 5-1).92–98 Several
studies on the kinetic mechanism of ALAS have been completed and have established
a proposed pathway by which glycine and succinyl-CoA combine to form δ-AL.99–102 In
the proposed mechanism (Figure 5-2), the decarboxylation of the bound glycine occurs
only after the Claisen condensation step and thus, may not be essential for catalysis.
We believe that this implies that there is potential for incorporating other amines that
lack carboxylate moieties as substitutes for glycine, which would significantly extend the
synthetic utility of ALAS. In support of this notion, it has been shown that methyl amine
binds to PLP and forms the external aldimine in ALAS.103 Formation of the external
aldimine by the binding of a substrate with PLP produces an increase in absorbance at
420 nm. The Ferreira group was able to prove that a methyl amine substrate would bind
to PLP and form the external aldimine by monitoring A420 absorbance. Whether other
amines can form the external aldimine and / or complete the catalytic cycle of ALAS
remains to be determined. All of these results taken together make ALAS an excellent
candidate for synthetic applications and protein engineering. Below, several positions
that have been subjected to site-directed mutagenesis are reviewed.
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Positions of Interest
Threonine 148
ALAS has been shown to be receptive towards protein engineering and mutants
that improved its substrate range and enhanced its catalytic efficiency have been
reported.104 Protein engineering of murine erythroid-specific ALAS (mALAS) has
revealed that mutations at position T148 can allow the enzyme to accept alternate
amino acids as substrates.100 Threonine at position 148 is conserved amongst the
structures of ALAS and the crystal structure of R. capsulatus ALAS shows that the
threonine at this position is located on a loop that directs its side-chain into the active
site (Figure 5-3).105 Furthermore, this side-chain lies near the glycine bound to PLP. The
steric bulk of the threonine side-chain at position 148 would make it difficult for any
substrate other than glycine to bind to PLP. Wild-type ALAS can accept serine in place
of glycine as a substrate, although the conversion is very poor. Mutagenesis studies
targeting T148 were carried out to improve the capacity of ALAS to accept serine.100 An
ALAS T148A mutant better accommodated serine, presumably because of the small
side-chain volume at this position. These results suggested to us that additional
mutations at position 148 might prove fruitful in further extending the substrate range of
ALAS.
Isoleucine 151
Isoleucine 151 is a second candidate for mutagenesis. The R. capsulatus ALAS
crystal structure shows that the side-chain of I151 also extends off the same loop as
T148 into the ALAS active site. Similar to T148, an isoleucine at position 151 is
generally conserved within the sequence of ALAS homologs.106 We hypothesized that
the side-chain of I151 partially dictates the position of the T148 side-chain. This would
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mean that residues at both positions 148 and 151 cooperatively impact amine substrate
specificity. For this reason, we chose position 151 for mutagenesis.
Arginine 85
ALAS accepts both β-hydroxybutyryl-CoA or acetoacetyl-CoA as substitutes for
succinyl-CoA, albeit at reduced relative rates (32% and 13%, respectively).100 Altering
R85 altered these substrate preferences and expanded the range of usable acyl-CoAs.
Ferreira mutated R21 in R. capsulatus ALAS (equivalent to R85 in murine ALAS) to
leucine and lysine This changed the enzyme’s preference from succinyl-CoA to butyryl-
and octanoyl-CoA.99 This result shows there is the potential to engineer this enzyme to
not only accept substitutes for the amino acid substrate (glycine), but also substitutes
for the acyl-CoA substrate (succinyl-CoA). This makes position R85 yet another
excellent candidate for mutagenesis.
The Glycine Loop
Amino acids T148 and I151 are located on an active site loop that directs both
residues into the proximity of the bound glycine external aldimine complex. Both of
these positions are of high interest for protein engineering. We intended to carry the
previous studies further and make full degenerate libraries at both positions T148 and
I151. Similarly, substitutions at position R85 have produced mALAS mutants that will
accept acyl compounds like butyryl-CoA and octanoyl-CoA as substitutes for succinyl-
CoA.99 We believe that this made position R85 another excellent position for
mutagenesis. Lastly, the loop that directs positions T148 and I151 is an area of interest
we wished to explore through randomization. Modifications along this loop may alter it in
subtle ways that would open up the area near the Pro-S hydrogen of a bound glycine,
thereby allowing amino acids with additional α-carbon functionality to bind. In an effort to
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effectively probe this region of the active site for interesting mutants, we wanted to
randomize the entire loop through cassette mutagenesis. Since the region of interest is
large, randomization in this manner would be the ideal way to tease out the mutants we
want.
Our long-term goal was to make ALAS mutants that could accept any amino acid
in addition to glycine and also accept a variety of acyl-CoA substrates. As ALAS
converts glycine and succinyl-CoA to δ-AL, it selectively deprotonates the Pro-R
hydrogen of glycine during a series of steps towards producing δ-AL. Since ALAS uses
a chirally-selective step in the mechanism, there is potential to use this enzyme to
produce chiral products (even though the normal product, δ-AL, is achiral). By
engineering ALAS to accept amino acid with chiral α-carbons, we hoped that the
products would maintain the optical purities of the reactants. Given the large range of
substrates to be explored, the significant extent of protein engineering planned, and the
laborious efforts required to purify mALAS mutants, we first required a “prescreening”
assay for our mutants that would reveal their catalytic (or lack of catalytic) activity
without requiring protein purification. Our plan was to prescreen transformants grown in
a δ-AL knockout host using the native reaction of glycine and succinyl-CoA to make δ-
AL, then further examine active variants with additional substrates using
spectrophotometric assays. The most promising transformants would be purified and
further characterized. This workflow was designed to cut down significantly the time
wasted by purifying nonfunctional mutants.
Project Overview
We assembled complete site-saturation mutagenesis libraries for each of the
three ALAS positions of greatest interest: R85, T148, and I151. We then prepared a
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template to be used for cassette mutagenesis at the glycine loop between positions
141-156. The original pGF23 vector was prepared with cassette mutagenesis in mind
but this vector did not target the areas of this loop as exclusively as we intended.104,107 A
silent mutation was introduced at position 161 that placed an Eco53kI restriction site at
this location. This site flanked the loop residues of interest, along with a pre-existing
AleI. This greatly simplified cassette mutagenesis that simultaneously targeted multiple
positions within the glycine loop.
We developed a prescreening assay for ALAS catalytic activity by derivatizing
the δ-AL-pyrrole with Ehrlich’s reagent. By reacting δ-AL with acetylacetone, a δ-AL-
pyrrole is made which is amenable to derivatization with Ehrlich’s reagent (Figure 5-4)
that forms an adduct with very strong absorbance at 553 nm (ϵ = 68,000 cm-1M-1) from
which the δ-AL concentration can be measured.108–110 Since this assay is robust and
can be used on a 96 well plate, it was ideal for screening several mutants.
We next developed an assay for measuring catalytic activity of mutants identified
from the prescreening step. One strategy was to quantitate acyl-CoA species using
reversed phase HPLC111 This would allow us to monitor our reactions that use acyl-CoA
substrates from the loss of these species and the appearance of free CoASH. We
validated this method by separating free CoASH from succinyl-CoA using HPLC (Figure
5-5).112,113 The advantage of this assay was that a variety of different acyl-CoA species
(not just succinyl-CoA) could be accommodated. The amino donor could also be varied
beyond glycine in this methodology. The disadvantage was that any acyl-CoA cleavage,
e.g., by spontaneous hydrolysis, added a background rate of acyl-CoA consumption.
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The second strategy for measuring ALAS catalytic activity was a coupled
spectrophotometric assay that used α-ketoglutarate dehydrogenase (α-KGD) to produce
succinyl-CoA in situ from CoA and α-ketoglutarate in an NAD+-dependent reaction. This
allowed the use of catalytic amounts of CoASH in the reactions and allowed the reaction
progress to be followed by measuring NADH formation by UV-Vis (Figure 5-6).114 This
assay could be used for amino substrates other than glycine, although it was limited to
succinyl-CoA as the second reactant.
The final analytical method we attempted to develop directly measured formation
of the final product of ALAS. We originally explored the nitrogen-derivatization reagent
phenylisothiocyanate (PITC, Edman’s reagent) prior to HPLC analysis of the reaction
mixture (Figure 5-7).115,116 Unfortunately, this reagent proved unsuccessful for δ-AL, so
we explored other methods based on GC-MS and MSTFA (Figure 5-8).117 Optimizing
this protocol to work with ALAS reactions would provide the final analytical tool to
evaluate ALAS mutants with either acyl-CoA or amino acid substitutes.
Results and Discussion
Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent
Our first task was to develop an assay that could detect δ-AL and other amino
products. The reaction of δ-AL with acetyl acetone produces a δ-AL-pyrrole. This
pyrrole will form a δ-AL-pyrrole Ehrlich derivative if subsequently reacted with Ehrlich’s
reagent. The resulting Ehrlich derivative is detectable at A553, with a ϵ = 68,000 cm-1M-1,
which allows quantification of the δ-AL produced in reactions with ALAS. We were able
to use this reaction successfully on products from reactions using ALAS to make δ-AL
from glycine and succinyl-CoA (Figure 5-9). This reaction was also successful when
using a plate assay containing several redundant reactions of δ-AL (Figure 5-10 and 5-
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11). It was our hope to develop a mid-level screening assay using this reaction to
screen through samples that succeeded during the E. coli HU 227 cell screening.
Preparation and Detection of Succinyl-CoA
Next, we set out to synthesize and detect succinyl-CoA. The standard method
involves mixing succinic anhydride and free CoASH with minimal stirring in an ice bath
for 30 min.112,113 Both succinyl-CoA product and residual CoASH can be separated and
quantitated by HPLC analysis. This provided us with a means to monitor the reaction
and ensure that it proceeded to completion.111 Further evidence that our synthesis of
succinyl-CoA was successful was obtained by reacting the reaction product with excess
hydroxylamine (Figure 5-12). Hydroxylamine is a strong nucleophile that cleaves
thioesters, yielding thiols and hydroxamic acids.118,119 Here, hydroxylamine formed
succinyl hydroxamic acid and regenerated CoASH, which could be followed by HPLC
(Figure 5-13 through 5-15). Iron(III)chloride was added to product to detect the
hydroxamic acid co-product, the solution’s color changed from yellow to dark red
indicating success. No spectral experiments were done on the product.
In Situ Succinyl-CoA Formation
While equimolar quantities of succinyl-CoA could be used for our studies, a
system requiring only catalytic quantities of CoASH would be much less expensive
when applied to a large number of samples. We therefore utilized an in situ succinyl-
CoA regeneration system that uses α-Ketoglutarate Dehydrogenase (α-KGD) to make
succinyl-CoA from CoASH and α-ketoglutarate.120 This system requires molar
equivalents of NAD+, α-ketoglutarate, and CoASH to produce succinyl-CoA. However,
since free CoASH is released as a by-product of δ-AL production by ALAS, the ALAS
reaction can be coupled to that of the α-KGD and provide a renewable supply of
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succinyl-CoA using only a catalytic quantity of CoASH. The coupling of CoA production
to NADH production is also useful in that NADH can be detected and quantified by A340
(ϵ = 6,220 (M-1cm-1).120 Thus, this technique provided a method to indirectly quantify the
succinyl-CoA to CoA conversion using a simple spectrophotometric assay. In reactions
without ALAS, the production of NADH stopped at a level corresponding to the
concentration of added CoASH, which is the limiting reagent in the absence of ALAS.
After this time, NADH production occurred at a greatly reduced rate, probably due to
spontaneous hydrolysis of succinyl-CoA under the reaction conditions (Figure 5-16).
HPLC analysis demonstrated that succinyl-CoA from CoASH and α-ketoglutarate under
these reaction conditions.
We tested the utility of this spectrophotometric assay by examining purified wild-
type mALAS and a series of mutants, generously provided by the Ferreira group.104 The
succinyl-CoA regeneration system was allowed to incubate for 15 min in order to
produce an initial amount of succinyl-CoA. ALAS was then added and the conversion of
NAD+ into NADH was monitored by A340. This set of experiments proved that the
conversion of succinyl-CoA to CoASH could be monitored for ALAS reactions (Figure 5-
17). The usefulness of this coupled assay cannot be understated. Since in addition to
providing a replenishing source of succinyl-CoA, it also provided an indirect method to
test any substrate that would be substituted for glycine. This is crucial since it is was our
goal to test a sizeable number of substrates against a myriad of mutants and several of
those are amino acid substitutes.
Detecting Amino Products Using PITC and MSTFA Derivatives
We next set out to find an effective way to detect the reaction products that ALAS
might make when other amines were substituted for glycine. The spectrophotometric
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assay described above only detects succinyl-CoA consumption, rather than directly
indicating C-C bond formation. It is also limited to reactions using succinyl-CoA as a
partner. We therefore sought a direct method for product detection and quantitation. We
first tried the classical method for detecting amino acids: PITC derivatization and HPLC
separation.115,116 We derivatized a set of amino acid standards using PITC. These
standards included: phenylalanine, leucine, alanine, glutamate, glycine, and δ-AL.
Separation of the PIT-amino acid derivatives by HPLC worked well for all standards
except PIT-δ-AL (Table 5-1). This was very unfortunate. Though we would ultimately
make products other than δ-AL, the products we would make would be similar to δ-AL,
so if this assay did not work on the parent compound, another assay was needed.
We next tried using MSTFA derivatization to enable GC-MS identification and
quantitation of ALAS products.117 Since all of the potential ALAS products would have a
primary amine with a labile proton, we hoped that they would be receptive to N-silylation
using MSTFA. This was indeed the case for glycine and δ-AL (Figure 5-18).
Unfortunately, while this methodology worked well with amino standards dissolved in 50
mM HEPES, pH 7.5, it failed when KPi buffer at any concentration was employed, and
so HEPES buffer was used. Though the reaction worked well enough for standards in
buffer solution (only HEPES) it was never successful at working with reactions using the
enzyme or with the other reaction conditions like the regeneration system. Using
mALAS reaction conditions greatly increased the number of unidentified peaks on the
chromatogram which further complicated identifying, much less quantifying, the TMS-
glycine and TMS-δ-AL peaks. Also, the glycerol from the stock solutions of both mALAS
and α-KGD may have sequestered water, which would be released once the solution
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was dissolved in the MSTFA solution and interfere with the derivatization reaction. This
method was thus never successfully used on reactions with mALAS. Thus, this assay
requires a significant amount of further optimization before it can be used to reliably
quantify mALAS products.
mALAS R85, T148 and I151 Site-Saturation Mutagenesis Libraries
We completed the cloning of complete, individual site-saturation mutagenesis
libraries of mALAS at positions R85, T148, and I151. All mutants for these libraries were
made individually and then used to transform E. coli HU 277 (an ALAS knockout
strain).104 We found concatameric primer inserts from the PCR were in the sequencing
of several these mutants. The artifacts almost exclusively contained a single reverse
primer insert, rather than the random mix of primer inserts that we observed during the
sequencing of the OYE 2.6 Y78 mutants mentioned in Chapter 2. Also, concatameric
mutants did not occur nearly as often as they did in the OYE 2.6 Y78 mutations. The
ratio of successful mutants to concatameric failures was: 4:1 for R85, 1:3 for T148, and
1:1 for T151. Making and sequencing redundant mutants allowed us to overcome this
problem. However, given the laborious requirements of making so many redundant
samples for such a large number mutants, and the difficulties of optimizing all of the
analytical methods, the protein purification was not successfully optimized. And as such,
optimizing the protein purification protocol is the next necessary step to screen our
mutants.
Experimental
General
Restriction endonucleases, Phusion Hot Start II High-Fidelity DNA Polymerase
and T4 DNA ligase were purchased from New England Biolabs. Primers were obtained
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from Integrated DNA Technologies. All other reagents were obtained from commercial
suppliers and used as received. Plasmids were purified on small scales by Wizard®
minicolumns (Promega Life Sciences) and on large scales using CsCl density gradient
ultracentrifugation. DNA sequencing was carried out by the University of Florida ICBR
using capillary fluorescence methods using standard protocols. LB medium contained 5
g/L Bacto-Yeast Extract, 10 g/L Bacto-Tryptone and 10 g/L NaCl.
Cloning
Construction of plasmid used to make ALAS libraries
The plasmid used as a template to make the ALAS libraries was pGF23 a CASS-
3 derivative prepared by Professor Gloria Ferreira, containing the gene for erythroid-
specific ALAS from mice (mALAS) (Appendix E, Figure E-5).107,121,122
Construction of ALAS libraries
The template used to make the mutants in all three ALAS libraries was pGF23. A
set of mutagenic primers containing a single codon replacement at either position R85,
T148, or I151 in ALAS was used to make each of the 19 mutants (primers are listed in
Appendix A, Table A-5). PCR was performed using 0.5 μL of 18 ng/μL template, 5 μL of
both 5 mM forward and reverse mutagenic primers, 1 μL of 10 mM dNTP mix, 10 μL of
5X HF Phusion® Hot start buffer, 28 μL of sterile water, and 0.5 μL of 2 U/μL Phusion®
Hot Start II DNA Polymerase for a total reaction volume of 50 μL. PCR was performed
using a MJ Mini® thermocycler from BioRad. PCR samples were run with an initial
denaturation step at 98°C for 30 s, then a subsequent 25 cycles of denaturation at 98°C
for 10 s, annealing at 64°C for 30 s, and an extension step at 72°C for 3 min 30 s, after
which the reactions were completed with a final extension step at 72°C for 7 min 30 s.
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All PCR samples were cleaned using Wizard® Plus SV Gel PCR Clean up kits by
Promega, using the manufacturer instructions. Samples were then incubated overnight
with two doses of 0.5 μL of 20 U/μL DpnI at 37°C to remove the parent template. DpnI is
an endonuclease that targets hemi methylated DNA and as such is ideal for removing
the superfluous template. The first dose of DpnI was added immediately after PCR
clean up and the second was added after 4 h of digestion. After DpnI digestion, samples
were cleaned using Wizard® Plus SV Gel PCR Clean up kits by Promega, using the
manufacturer instructions.
Following the PCR work up, PCR samples were transformed by electroporation
into ETB cells using a Gene Pulser® from BioRad. Electroporation was carried out with
2 - 6 μL of PCR sample and 50 µL of ETB cells under 2.5 kV. Electroporated samples
were incubated in 600 μL of SOC media at 37°C for 1 h. Cells were then plated onto LB-
amp agar plates and grown at 37°C for 36 h. Transformant cells were sequenced to
verify that the desired mutation was present at the ICBR complex using Sanger
sequencing. Plasmids that were verified by sequencing to contain the desired mutant
were then transformed in an expression strain. Transformation was done using
electroporation under 2.5 kV with 4 μL of 10 ng/μL of plasmid with 80 μL of E. coli HU
227 electrocompetent cells. Electroporated samples were incubated in 600 μL of SOC
medium at 37°C for 45 min. Cells were then plated onto LB-amp agar plates and grown
at 37°C overnight. After mutations were verified by sequencing, transformants were
assembled into a 96 well plate. Transformants were grown in 600 μL of LB-amp in a 96
well plate overnight to reach saturation. The library was completed with the transfer of
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120 μL of saturated cultures into a new 96 well plate containing 30 μL of 80% glycerol
which brought the final concentration of glycerol to 15%.
Preparation and Detection of Succinyl-CoA
Succinyl-CoA was prepared using succinic anhydride and CoA. CoA was from
Sigma-Aldrich. Succinic anhydride was thoroughly ground using a beaker and wax
paper. An adequate amount (between 1/2 and 1/3 a centimeter) of ground succinic
anhydride was loaded into a yellow pipette tip by pressing into succinic anhydride.
Succinic anhydride was then transferred into a 100 μL aqueous solution containing 20
mM CoA. 800 μL of ice cold D.I. water was added to the solution bringing the CoA to a
concentration of 22 mM. The solution was then mixed every 5 min using a pipette while
in ice bath for 30 min. After 30 min 100 μL of 1 M HCO3 was added bringing solution to
final volume of 1 mL with 100 mM HCO3, and 2 mM succinyl-CoA. Succinyl-CoA was
immediately stored on ice and used for experiments.
Reactions were then verified by HPLC and separated on a 150 × 4.6 mm Synergi
Hydro-RP 80 Å column using reverse phase HPLC. Solvent A was 50 mM NaPi buffer
with a pH of 5.0 and Solvent B was 50 mM NaPi with a pH of 5.0 and 20 % of
acetonitrile. The flow rate was 1 mL/min and samples were monitored by UV
absorbance simultaneously at both 220 and 260 nm. Initial conditions (3% B) were
maintained for 2.5 min; then a linear increase to 18% B over 5 min was immediately
followed by a linear increase to 28% B over 3.5 min, then a linear increase to 90% B
over 10 min. After a 5 min hold at 90% B, a linear decrease to 3% B over 3 min was
followed by a 5 min hold at the initial conditions (3% B) (HPLC method is listed as
LMM.Meth in Appendix D).123 CoA had eluted at 14.9 min, succinyl-CoA eluted at 15.5
min, and caffeine eluted at 24.3 min (caffeine was used as an internal standard). The
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CoA peak was verified by spiking reactions with 20 mM CoA. A succinyl-CoA standard
was far too expensive to use, so an indirect method was needed to verify the succinyl-
CoA peak. The first method used to verify the succinyl-CoA was reaction with
hydroxylamine. Hydroxylamine can be used as a nucleophile to break thiol esters into
thiols and hydroxamic acids. In the case of this reaction, it displaces the succinyl adduct
forming succinyl-hydroxamic acid and regenerating CoA. By monitoring the
disappearance of the presumptive succinyl-CoA peak and the increase of the
established CoA peak on HPLC we were able to verify the succinyl-CoA peak’s position.
Further verification of the succinyl-CoA peak was done using the succinyl-CoA
regeneration system. Aliquots were taken from a reaction using the succinyl-CoA
regeneration system after the NADH production had topped off and analyzed by HPLC.
The major product peak from the succinic anhydride peak matched the major product
peak for the regeneration system.
Succinyl-CoA Regeneration System
A 2 mL reaction mix was prepared using 20 mM HEPES, 3.0 mM MgCl2,1.0 mM
α-ketoglutarate, 1.0 mM NAD+, 250 μM thiamine pyrophosphate, 100 mM amino acid
substrate, 20 μM CoA, and 2.9 U/mL α-KGD. 1 mL of this solution was aliquoted out to
be used as a blank and the other 1 mL was saved to be used for the reaction with
enzyme. Reactions were incubated at room temperature for 20 min to allow an initial
amount of succinyl-CoA to be made by α-KGD before being analyzed by
spectrophotometry. Reactions with enzyme included 10 µL of mALAS from a glycerol
stock solution (concentrations were not determined). Reactions were analyzed using a 2
mL quartz cuvette with a 1 cm path length in an Agilent 8453 UV-Vis spectrophotometer
at 30°C. Reactions were initiated by the addition of the mALAS. NADH can be detected
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and quantified by A340, with a ϵ = 6,220 (M cm)-1.120 Reactions were observed over a
course of 20 min. Aliquots of the reaction blank, solutions that were absent of any
mALAS, were separated by HPLC to verify that succinyl-CoA had been made.
Detecting δ-AL-Pyrrole Compounds with Ehrlich’s Reagent
2 mL of 0.2 mM δ-AL was combined with 5 mL of 1 M acetic acid buffer at pH
4.6. Then 200 μL of acetyl acetone was added to the solution. A sufficient quantity of
acetic acid buffer was added to bring the final volume to 10 mL with a concentration of
20 μM δ-AL. The reaction was then submerged in a boiling water bath for 10 min. 1 mL
aliquots of 20 μM, 10 μM, & 5 μM δ-AL-pyrrole were prepared from this stock solution
using 1 M acetic acid buffer to bring the volumes to 1 mL. Ehrlich’s reagent was
prepared by dissolving 100 mg of Ehrlich’s reagent in 5 mL of 2 N Perchloric acid. 1 mL
of Ehrlich’s reagent was combined with each of the 1 mL δ-AL-pyrrole aliquots.
Reactions with the Ehrlich’s reagent were scanned with the spectrophotometer exactly 1
min after addition of the Ehrlich’s reagent. Reactions were monitored at a ʎ = 553 nm
using a 2 mL quartz cuvette in an Agilent 8453 UV-Vis spectrophotometer over 10 min
at room temperature. The resulting Ehrlich derivative is detectable at A553, with a ϵ =
68,000 (cm M)-1.
Reactions testing δ-AL production using mALAS began with the same reaction
conditions mentioned in the succinyl-CoA regeneration system section. A 100 μL aliquot
of an overnight reaction of succinyl-CoA with glycine using mALAS was combined with
5 mL of 1 M acetic acid pH 4.6. 100 µL of acetyl acetone was added to this reaction
mixture and mixed by inversion. The reaction mixture was placed in a boiling water bath
for 10 min. Ehrlich’s reagent was prepared the same way mentioned previously. 1 mL of
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Ehrlich’s reagent was combined with 1 mL of reaction mixture and scanned with
spectrophotometer using the same conditions mentioned above.
Reactions testing δ-AL production using mALAS with a plate assay began with
the same reaction conditions mention in the succinyl-CoA regeneration system section.
200 μL of this reaction mixture was aliquoted into 8 wells of a micro titer plate and
incubated at room temperature overnight. Then a master mix containing 40 μL of acetyl
acetone and 2 mL of 1 M acetic acid pH 4.6 was prepared. 200 μL of the acetyl acetone
master mix was aliquoted into a 96 well plate. Next, 3 μL of this reaction mixture was
transferred to the 96 well plate with the acetyl acetone aliquots. The entire plate was
then submerged in a boiling water bath for 10 min. Ehrlich’s reagent was then prepared
as previously mentioned. 150 μL of Ehrlich’s reagent was aliquoted into the δ-AL-pyrrole
reaction. Reactions were scanned with a SpectraMax 190 Microplate Reader from
wavelengths between 460-780 nm.
Derivatizing Amino Acids Using PITC
Using 10 μL of a 10 mM amino acid solution was dried using a Speed Vac to
remove solvent. Dry sample pellets were dissolved in 100 μL of a coupling buffer
containing 50% acetonitrile, 25% pyridine, 10% triethylamine, 15% D.I. water. Samples
were then dried using a Speed Vac to remove solvent a second time. Dry sample
pellets were dissolved in 100 μL of the coupling buffer a second time. After
reconstitution in coupling buffer, 5 μL of PITC was added to each sample and they were
incubated at room temperature for 5 min. Samples were then dried using a Speed Vac
to remove solvent for a third time. Samples were then dissolved in 250 μL of 50 mM
NH4 Acetate pH 6.8 (solvent A for the HPLC separation).
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Detecting PITC Amino Acid Derivatives by HPLC
PITC amino acid derivatives can be separated using 150 × 4.6 mm Synergi
Hydro-RP 80 Å column using reverse phase HPLC. Solvent A was 50 mM NH4 Acetate
pH 6.8 and solvent B was 100 mM NH4 Acetate pH 6.8 with 45% acetonitrile, and 10%
methanol. Flow rate was 1 mL/min. Program began with 100 % solvent A with an
immediate increase of solvent B to 15% over 15 min, followed by an increase of solvent
B to 50% over 15 min, followed by an increase of solvent B to 100% over 4 min at which
the program stayed for 3 min, this was followed by a decrease of solvent B to 0% over 3
min at which the program remained for 10 min (HPLC method is listed as RWP2.Meth in
Appendix D). PITC amino acid derivatives eluted near 33.9 min for Phe, 31.2 min for
Leu, 6.6 min for Glu, 17.3 min for Ala, 12.0 min for Gly, and δ-AL was never determined.
Derivatizing Amino Acids Using MSTFA
Standards of δ-AL and glycine were used for derivatization with MSTFA. δ-AL
and glycine were dissolved in either D.I. water or 100 mM HEPES pH 7.5. A Speed Vac
was used to dry the samples until all solvent was removed and a pellet was formed.
Depending on the volume of the samples (typically samples were between 100-250 μL)
removing the solvent would take between 1-2 h. The pellets were resuspended with 50
μL of an 80:20 methanol / water mix. Solvent was then removed a second time using a
Speed Vac until a pellet forms again. The pellets were then resuspended again with 50
μL of methylene chloride, adding one drop at a time (usually between 5-6 drops). The
solvent was removed a third time using a Speed Vac until a pellet formed yet again.
After this succession of solvent switches was completed, 1 drop of pyridine was added
to the pellets. Next, 50 μL of MSTFA was added to the samples. Samples were placed
in a microfuge tube holder and secured into a 37°C shaker with 250 rpm of agitation for
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30 min. After samples returned to room temperature they were mixed by vortexing and
the immediately analyzed by GC-MS.
Detecting MSTFA Amino Acid Derivatives Using GC-MS
TMS-amino acid derivatives can be detected by GC-MS using a DB-17 column
(0.25 mm x 30 m). The temperature program used to detect TMS-amino acid derivatives
began with an initial temperature of 60°C for 5 min, followed by an increase at 10°C/min
to a temperature of 195°C at which the program remained for 10 min (GC method is
listed as SEF.Meth in Appendix D). TMS-glycine eluted near 9.8 min and TMS-δ-AL
eluted near 16.1 min.
Conclusions
Though we were able to make all three mALAS libraries we were not able to
screen them. We were not able to successfully overexpress and purify mALAS. Also,
we were not able to detect PITC or MSTFA derivatized δ-AL products using our reaction
conditions by GC or HPLC. These are both areas which require further optimization.
Should we never successfully optimize the MSTFA assay to work with our reaction
conditions, or devise a new method for directly detecting our products, we do have other
options. We were able to get several analytical methods to work using the mALAS
reaction conditions during this project. One such method was the spectrophotometric
assay using the α-KGD regeneration system. This assay is the most reliable method we
have for detecting mALAS activity for amino acid substitutes for glycine. So if the
MSTFA protocol cannot be successfully optimized then this spectrophotometric assay
would be our best method for detecting products when we use substitutes for glycine.
However, though we can substitute for glycine with this assay, we cannot use it with any
substrate other than succinyl-CoA, and we would like to substitute different acyl-CoA
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substrates for succinyl-CoA. So should it become necessary, we can separate acyl-CoA
substrates from CoA by HPLC. Using this method we could obtain the ratios of
substrates to products. Of course screening mutants with either method would require
us to successfully purify the mALAS mutants. And as such, it would be the most
immediate protocol we would need to optimize.
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Table 5-1. Retention times of PIT-amino acid derivative standards from HPLC.
Amino Acid Standard HPLC retention times of PIT-amino acid standards from assay (min)
HPLC retention times PIT-amino acid standards from the literature (min)
PIT-Phenylalanine 33.1 32.0
PIT-Alanine 17.3 15.5
PIT-Glycine 12.0 10.0
PIT-Glutamate 6.6 4.5
PIT-Leucine 31.2 30.0
PIT-δ-AL Not detected Not reported
210
Figure 5-1. The reaction of glycine and succinyl-CoA to make δ-AL using ALAS as a
catalyst.
211
Figure 5-2. The proposed mechanism of ALAS.
212
Figure 5-3. The active site of ALAS from R. capsulatus with glycine bound to PLP (2BWP). The hydrophobic positions of interest for protein engineering are shown in orange and the values in parenthesis are the positions in the mALAS structure. The glycine loop is shown in blue and glycine bound to PLP is shown in yellow.
Figure 5-4. The active site of ALAS from R. capsulatus with succinyl-CoA (2BWO). The arginine position of interest for protein engineering is shown in orange.
213
Figure 5-5. Reaction scheme of the derivatizing of δ-AL-pyrrole with Ehrlich’s reagent.
Figure 5-6. Reaction scheme of succinic anhydride with CoA to make succinyl-CoA.
214
Figure 5-7. The coupling of ALAS production of CoA from succinyl-CoA to α-Ketoglutarate Dehydrogenase production of NADH from NAD+.
215
Figure 5-8. Derivatization of amino acids using PITC.
Figure 5-9. Derivatization of amino acids using MSTFA.
Figure 5-10. The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent. These results were obtained from reactions using mALAS. The reaction that ran overnight using mALAS gave three times the product as the reaction that was run for 1 h. This chart also shows that it takes only about 5 mins for the reaction of δ-AL-pyrrole with Ehrlich’s reagent to go to completion.
216
Figure 5-11. The results for the reaction of δ-AL-pyrrole with Ehrlich’s reagent using a plate reader. Eight redundant reactions were run overnight with mALAS wt. using the regeneration system. Reactions were scanned with a SpectraMax 190 Microplate Reader from wavelengths between 460-780 nm. The Ehrlich’s derivative absorbs very strongly near 553 nm. Our sample with δ-AL-pyrrole is slightly blue shifted. All samples gave identical results, this figure shows the results for one sample.
217
Figure 5-12. The reaction of succinyl-CoA with hydroxyl amine to displace the CoA.
Figure 5-13. HPLC results from the reaction of succinic anhydride and CoA to make succinyl-CoA. Succinyl-CoA Rt = 15.5 mins, CoA Rt = 14.9 mins, & caffeine Rt = 24.3 mins. This chromatograph shows our reaction to make Succinyl-CoA has gone to completion.
218
Figure 5-14. HPLC results from the synthesis of succinyl-CoA from succinic anhydride and CoA co-eluted with 10x CoA. Succinyl-CoA Rt = 15.5 mins, CoA Rt = 14.9 mins, & caffeine Rt = 24.3 mins. This chromatograph shows our completed reaction spiked with 10x CoA standard and verifies the location of CoA.
219
Figure 5-15. HPLC results from the reaction of succinyl-CoA with hydroxylamine. Succinyl-CoA Rt = 15.5 mins, & CoA Rt = 14.9 mins. Using an aliquot of our completed succinyl-CoA reaction, we ran the hydroxyl amine to displace the CoA. What this chromatograph shows is that the succinyl-CoA has been completely displaced and the CoA peak reappears.
220
Figure 5-16. Results of the succinyl-CoA regeneration system. Α-KGD takes less than a minute to convert all the CoA into succinyl-CoA. At this point the NADH concentration detected is equivalent to the maximum concentration of CoA (NADH maximum which is the orange line). Hydrolysis of succinyl-CoA back to CoA in solution is what causes the slow increase afterwards.
221
Figure 5-17. Results of reactions from the succinyl-CoA regeneration system using mALAS and mALAS R433K. Both mALAS Wt and the mALAS R433K batches covert glycine to δ-AL and that detection is indirectly monitored using the α-KGD regeneration system.
222
Figure 5-18. Results of amino acid derivatization with MSTFA. These results are from the standards of mixtures of glycine and δ-AL. Neither enzyme, buffer, nor any other component was present in these standards.
223
APPENDIX A LIST OF PRIMERS
Chapter 1 Primers
Table A-1. List of mutagenic primers for Chapter 1.
Mutation Sequence
Y196X Fwd 5’-GTG CTA ACG GTN NKT TGT TAA ACC AGT TCT TGG ACC CTC A-3’
Y196X Rev 5’-TGG TTT AAC AAM NNA CCG TTA GCA CTG TGA ATT TCA ACA C-3’
Y196W Fwd 5’-GTG CTA ACG GTT GGT TGT TAA ACC AGT TCT TGG ACC CTC A-3’
Y196W Rev 5’-TGG TTT AAC AAC CAA CCG TTA GCA CTG TGA ATT TCA ACA C-3’
224
Chapter 2 Primers
Table A-2. List of mutagenic and sequencing primers for Chapter 2.
Mutation Sequence
Y78T Fwd 5’-AAG CCT CTG GTA CTG AAG GTG CTG CTC CAG GTA TTT GGA C-3’
Y78T Rev 5’-GCA GCA CCT TCA GTA CCA GAG GCT TGA GGA GAG ACA AAA G-3’
Y78V Fwd 5’-AAG CCT CTG GTG TTG AAG GTG CTG CTC CAG GTA TTT GGA C-3’
Y78V Rev 5’-GCA GCA CCT TCA ACA CCA GAG GCT TGA GGA GAG ACA AAA G-3’
Y78XKST Fwd 5’-AAG CCT CTG GTK STG AAG GTG CTG CTC CAG GTA TTT GGA C-3’
Y78XKST Rev 5’-GCA GCA CCT TCA TAA CCA GAG GCT TGA GGA GAG ACA AAA G-3’
I113A Fwd 5’-CAA CCC AGT TGG CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113A Rev 5’-CTT CCC AAA AAA GCC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113C Fwd 5’-CAA CCC AGT TGT GTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113C Rev 5’-CTT CCC AAA AAA CAC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113G Fwd 5’-CAA CCC AGT TGG GTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113G Rev 5’-CTT CCC AAA AAA CCC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113S Fwd 5’-CAA CCC AGT TGT CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113S Rev 5’-CTT CCC AAA AAA GAC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113T Fwd 5’-CAA CCC AGT TGA CTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113T Rev 5’-CTT CCC AAA AAA GTC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113V Fwd 5’-CAA CCC AGT TGG TTT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113V Rev 5’-CTT CCC AAA AAA ACC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
I113XKST Fwd 5’-CAA CCC AGT TGK STT TTT TGG GAA GGG TTG CAG ATC CAG C-3’
I113XKST Rev 5’-CTT CCC AAA AAA SMC AAC TGG GTT GAA ACG AAA GAA CCG T-3’
F247X Fwd 5’-CAT GGG CTA CTN NKC AAA ACA TGA AGG CTC ACA AGG ACA C-3’
F247X Rev 5’-TTC ATG TTT TGM NNA GTA GCC CAT GGA GAG ATT CTG ATA C-3’
OYE 2.6 Seq 5’-TCC AGC AAG TAT ATA GCA TGG CCT-3’
225
Chapter 3 Primers
Table A-3. List of mutagenic and sequencing primers for Chapter 3.
Mutation Sequence
E41X Fwd 5’-TTA GAG CTT TAN NKG ACC ACA CTC CTT CTG ATT TGC AAT T-3’
E41X Rev 5’-GGA GTG TGG TCM NNT AAA GCT CTA AAT CTA GTA GTT GGT G-3’
D141X Fwd 5’-CTT ATG AAA GTN NKG CCG CTA AAG AAG CTG CCG AAG CAG T-3’
D141X Rev 5’-TCT TTA GCG GCM NNA CTT TCA TAA GTA GCA GAG GCA GAA A-3’
E145X Fwd 5’-ATG CCG CTA AAN NKG CTG CCG AAG CAG TTG GTA ACC CTG T-3’
E145X Rev 5’-GCT TCG GCA GCM NNT TTA GCG GCA TCA CTT TCA TAA GTA G-3’
K330X Fwd 5’- CTC CAG AGT TCN NKA CAT TGA AGG AAG ATA TCG CTG ACA A-3’
K330X Rev 5’-TCC TTC AAT GTM NNG AAC TCT GGA GCA TCG TAG GAG TAG T-3’
I214X Fwd 5’-ACG GTG GAT CCN NKG AGA ACA GAG CCA GGT TAA TTC TTG A-3’
I214X Rev 5’-GCT CTG TTC TCM NNG GAT CCA CCG TAT TCA TCA GTT CTT T-3’
W244X Fwd 5’-GAA TCT CTC CAN NKG CTA CTT TCC AAA ACA TGA AGG CTC A-3’
W244X Rev 5’-TGG AAA GTA GCM NNT GGA GAG ATT CTG ATA CCA ATC TTG T-3’
L260X Fwd 5’- CTG TTC ACC CAN NKA CTA CTT TCT CTT ACT TGG TCC ACG A -3’
L260X Rev 5’-GAG AAA GTA GTM NNT GGG TGA ACA GTG TCC TTG TGA GCC T-3’
F307X Fwd 5’-GTG ACA ACG AAN NKG TCT CCA AGA TCT GGA AGG GTG TTA T-3’
F307X Rev 5’-ATC TTG GAG ACM NNT TCG TTG TCA CCA GCT TGG TCT TCT T-3’
I311X Fwd 5’-TTG TCT CCA AGN NKT GGA AGG GTG TTA TCT TGA AGG CAG G-3’
I311X Rev 5’-ACA CCC TTC CAM NNC TTG GAG ACA AAT TCG TTG TCA CCA G-3’
S388P Fwd 5’-TTT CTA TGG ATC CGG AAG AGG TTG ATA AAG AAT TAG AAA T-3’
S388P Rev 5’-TCA ACC TCT TCC GGA TCC ATA GAA AAG GTA TTG TAA CCA G-3’
S388I Fwd 5’-TTT CTA TGG ATA TTG AAG AGG TTG ATA AAG AAT TAG AAA T-3’
S388I Rev 5’-TCA ACC TCT TCA ATA TCC ATA GAA AAG GTA TTG TAA CCA T-3’
S388N Fwd 5’-TTT CTA TGG ATA ATG AAG AGG TTG ATA AAG AAT TAG AAA T-3’
S388N Rev 5’-TCA ACC TCT TCA TTA TCC ATA GAA AAG GTA TTG TAA CCA T-3’
S388Y Fwd 5’-TTT CTA TGG ATT ATG AAG AGG TTG ATA AAG AAT TAG AAA T-3’
S388Y Rev 5’-TCA ACC TCT TCA TAA TCC ATA GAA AAG GTA TTG TAA CCA T-3’
OYE 2.6 Seq 5’-TCC AGC AAG TAT ATA GCA TGG CCT-3’’
226
Chapter 4 Primers
Table A-4. List of mutagenic and sequencing primers for Chapter 4.
Mutation Sequence
Y389Y Fwd 5’-ACT ACC CAA CAT ACG AAG AGG CAG TAG ATT TAG GTT GGA A-3’
Y389Y Rev 5’-ACT GCC TCT TCG TAT GTT GGG TAG TCG GTA TAA CCT TCC G-3’
NdeI(-) Fwd 5’-AAG GAG ATA TAC AAA TGC CCA TGG ATA TCG GAA TTA ATT C-3’
NdeI(-) Rev 5’-TCC ATG GGC ATT TGT ATA TCT CCT TCT TAA AGT TAA ACA A-3’
NdeI(+)Fwd 5’-ATC CGA ATT CGC ATA TGC CAT TTG TAA AAG GTT TTG AGC C-3’
NdeI(+)Rev 5’-ACA AAT GGC ATA TGC GAA TTC GGA TCC GAA TTA ATT CCG A-3’
F296S Fwd 5’-GTA ACT AAC CCA TCC TTG ACT GAA GGG GAG GGT GAA TAC G-3’
F296S Rev 5’-CCT TCA GTC AAG GAT GGG TTA GTT ACA CGA GGT TCA ACC A-3’
W116A Fwd 5’-GGG TAC AAC TTG CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116A Rev 5’-CAG CCT AAA GAA GCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116C Fwd 5’-GGG TAC AAC TTT GTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116C Rev 5’-CAG CCT AAA GAA CAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116D Fwd 5’-GGG TAC AAC TTG ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116D Rev 5’-CAG CCT AAA GAA TCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116E Fwd 5’-GGG TAC AAC TTG AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116E Rev 5’-CAG CCT AAA GAT TCA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116F Fwd 5’-GGG TAC AAC TTT TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116F Rev 5’-CAG CCT AAA GAC CAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116G Fwd 5’-GGG TAC AAC TTG GGT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116G Rev 5’-CAG CCT AAA GAA CGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116H Fwd 5’-GGG TAC AAC TTC ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116H Rev 5’-CAG CCT AAA GAA TGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116I Fwd 5’-GGG TAC AAC TTA TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116I Rev 5’-CAG CCT AAA GAA ATA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116K Fwd 5’-GGG TAC AAC TTA AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116K Rev 5’-CAG CCT AAA GAT TTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116L Fwd 5’-GGG TAC AAC TTC TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116L Rev 5’-CAG CCT AAA GAA AGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116M Fwd 5’-GGG TAC AAC TTA TGT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116M Rev 5’-CAG CCT AAA GAC ATA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116N Fwd 5’-GGG TAC AAC TTA ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116N Rev 5’-CAG CCT AAA GAA TTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116P Fwd 5’-GGG TAC AAC TTC CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
227
Table A-4. Continued
Mutation Sequence
W116P Rev 5’-CAG CCT AAA GAA GGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116Q Fwd 5’-GGG TAC AAC TTC AAT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116Q Rev 5’-CAG CCT AAA GAT TGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116R Fwd 5’-GGG TAC AAC TTC GTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116R Rev 5’-CAG CCT AAA GAA CGA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116S Fwd 5’-GGG TAC AAC TTT CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116S Rev 5’-CAG CCT AAA GAA GAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116T Fwd 5’-GGG TAC AAC TTA CTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116T Rev 5’-CAG CCT AAA GAA GTA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116V Fwd 5’-GGG TAC AAC TTG TTT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116V Rev 5’-CAG CCT AAA GAA ACA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
W116Y Fwd 5’-GGG TAC AAC TTT ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
W116Y Rev 5’-CAG CCT AAA GAA TAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
OYE 3 seq 5’-AGC GTT TGG CCT TTG TGC ACC TCG-3’
228
Chapter 5 Primers
Table A-5. List of mutagenic and sequencing primers for Chapter 5.
Mutation Sequence
R85A Fwd 5’-ACC ACA CCT ACG CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85A Rev 5’-GTC TTG AAC ACA GCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85C Fwd 5’-ACC ACA CCT ACT GTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85C Rev 5’-GTC TTG AAC ACA CAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85D Fwd 5’-ACC ACA CCT ACG ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85D Rev 5’-GTC TTG AAC ACA TCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85E Fwd 5’-ACC ACA CCT ACG AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85E Rev 5’-GTC TTG AAC ACT TCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85F Fwd 5’- ACC ACA CCT ACT TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3
R85F Rev 5’- GTC TTG AAC ACA AAG TAG GTG TGG TCC TGT TTC TTC TCC A-3
R85G Fwd 5’- ACC ACA CCT ACG GGG TGT TCA AGA CTG TGA ATC GTT GGG C-3
R85G Rev 5’-GTC TTG AAC ACC CCG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85H Fwd 5’- ACC ACA CCT ACC ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85H Rev 5’- GTC TTG AAC ACA TGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85I Fwd 5’-ACC ACA CCT ACA TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85I Rev 5’-GTC TTG AAC ACA ATG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85K Fwd 5’-ACC ACA CCT ACA AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85K Rev 5’-GTC TTG AAC ACT TTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85L Fwd 5’-ACC ACA CCT ACC TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85L Rev 5’-GTC TTG AAC ACA AGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85M Fwd 5’-ACC ACA CCT ACA TGG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85M Rev 5’-GTC TTG AAC ACC ATG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85N Fwd 5’-ACC ACA CCT ACA ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85N Rev 5’-GTC TTG AAC ACA TTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85P Fwd 5’-ACC ACA CCT ACC CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85P Rev 5’-GTC TTG AAC ACA GGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85Q Fwd 5’-ACC ACA CCT ACC AAG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85Q Rev 5’-GTC TTG AAC ACT TGG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85S Fwd 5’-ACC ACA CCT ACA GTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85S Rev 5’-GTC TTG AAC ACA CTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85T Fwd 5’-ACC ACA CCT ACA CTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85T Rev 5’-GTC TTG AAC ACA GTG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85V Fwd 5’-ACC ACA CCT ACG TTG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
229
Table A-5. Continued
Mutation Sequence
R85V Rev 5’-GTC TTG AAC ACA ACG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85W Fwd 5’-ACC ACA CCT ACT GGG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85W Rev 5’-GTC TTG AAC ACC CAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
R85Y Fwd 5’-ACC ACA CCT ACT ATG TGT TCA AGA CTG TGA ATC GTT GGG C-3’
R85Y Rev 5’-GTC TTG AAC ACA TAG TAG GTG TGG TCC TGT TTC TTC TCC A-3’
T148A Fwd 5’-GAG CTG GGG GCG CTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148A Rev 5’-GAG ATA TTG CGA GCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148C Fwd 5’-GAG CTG GGG GCT GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148C Rev 5’-GAG ATA TTG CGA CAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148D Fwd 5’-GAG CTG GGG GCG ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148D Rev 5’-GAG ATA TTG CGA TCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148E Fwd 5’-GAG CTG GGG GCG AGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148E Rev 5’-GAG ATA TTG CGC TCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148F Fwd 5’-GAG CTG GGG GCT TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148F Rev 5’-GAG ATA TTG CGA AAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148G Fwd 5’-GAG CTG GGG GCG GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148G Rev 5’-GAG ATA TTG CGA CCG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148H Fwd 5’-GAG CTG GGG GCC ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148H Rev 5’-GAG ATA TTG CGA TGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148I Fwd 5’-GAG CTG GGG GCA TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148I Rev 5’-GAG ATA TTG CGA ATG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148K Fwd 5’-GAG CTG GGG GCA AAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148K Rev 5’-GAG ATA TTG CGT TTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148L Fwd 5’-GAG CTG GGG GCC TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148L Rev 5’-GAG ATA TTG CGA AGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148M Fwd 5’-GAG CTG GGG GCA TGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148M Rev 5’-GAG ATA TTG CGC ATG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148N Fwd 5’-GAG CTG GGG GCA ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148N Rev 5’-GAG ATA TTG CGA TTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148P Fwd 5’-GAG CTG GGG GCC CTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148P Rev 5’-GAG ATA TTG CGA GGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148Q Fwd 5’-GAG CTG GGG GCC AAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148Q Rev 5’-GAG ATA TTG CGT TGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148R Fwd 5’-GAG CTG GGG GCA GAC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148R Rev 5’-GAG ATA TTG CGT CTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
230
Table A-5. Continued
Mutation Sequence
T148S Fwd 5’-GAG CTG GGG GCA GTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148S Rev 5’-GAG ATA TTG CGA CTG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148V Fwd 5’-GAG CTG GGG GCG TTC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148V Rev 5’-GAG ATA TTG CGA AGG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148W Fwd 5’-GAG CTG GGG GCT GGC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148W Rev 5’-GAG ATA TTG CGC CAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
T148Y Fwd 5’-GAG CTG GGG GCT ATC GCA ATA TCT CAG GTA CCA GCA AGT T-3’
T148Y Rev 5’-GAG ATA TTG CGA TAG CCC CCA GCT CCA GCT CCA TGA TTC T-3’
I151A Fwd 5’-GCA CTC GCA ATG CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151A Rev 5’-CTG GTA CCT GAG GCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151C Fwd 5’-GCA CTC GCA ATT GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151C Rev 5’-CTG GTA CCT GAG CAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151D Fwd 5’-GCA CTC GCA ATG ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151D Rev 5’-CTG GTA CCT GAG TCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151E Fwd 5’-GCA CTC GCA ATG AGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151E Rev 5’-CTG GTA CCT GAC TCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151F Fwd 5’-GCA CTC GCA ATT TCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151F Rev 5’-CTG GTA CCT GAG AAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151G Fwd 5’-GCA CTC GCA ATG GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151G Rev 5’-CTG GTA CCT GAG CCA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151H Fwd 5’-GCA CTC GCA ATC ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151H Rev 5’-CTG GTA CCT GAG TGA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151K Fwd 5’-GCA CTC GCA ATA AGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151K Rev 5’-CTG GTA CCT GAC TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151M Fwd 5’-GCA CTC GCA ATA TGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151M Rev 5’-CTG GTA CCT GAC ATA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151N Fwd 5’-GCA CTC GCA ATA ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151N Rev 5’-CTG GTA CCT GAG TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151P Fwd 5’-GCA CTC GCA ATC CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151P Rev 5’-CTG GTA CCT GAG GGA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151Q Fwd 5’- GCA CTC GCA ATA ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151Q Rev 5’- CTG GTA CCT GAG TTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151R Fwd 5’- GCA CTC GCA ATA GGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151R Rev 5’-CTG GTA CCT GAC CTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151S Fwd 5’-GCA CTC GCA ATA GCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
231
Table A-5. Continued
Mutation Sequence
I151S Rev 5’-CTG GTA CCT GAG CTA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151T Fwd 5’-GCA CTC GCA ATA CCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151T Rev 5’-CTG GTA CCT GAG GTA TTG CGA GTG CCC CCA GCT CCA GCTC-3’
I151V Fwd 5’-GCA CTC GCA ATG TCT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151V Rev 5’-CTG GTA CCT GAG ACA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151W Fwd 5’-GCA CTC GCA ATT GGT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151W Rev 5’-CTG GTA CCT GAC CAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
I151Y Fwd 5’-GCA CTC GCA ATT ACT CAG GTA CCA GCA AGT TTC ATG TGG A-3’
I151Y Rev 5’-CTG GTA CCT GAG TAA TTG CGA GTG CCC CCA GCT CCA GCT C-3’
L161L Fwd 5’-GGG TAC AAC TTT ATT CTT TAG GCT GGG CAT CCT TCC CAG A-3’
L161L Rev 5’-CAG CCT AAA GAA TAA AGT TGT ACC CAC GCG AAC GAC TGA C-3’
232
APPENDIX B
MUTAGENIC PLASMIDS
Table B-1. Mutagenic plasmids used in this study.
Plasmid Parent Mutation Description
pET3b T-7 promoter, and AmpR
pET3b-OYE pET3b OYE 1 gene OYE 1 non-tagged protein
pET30a T-7 promoter, His-Tag, and KanR
pET30a-OYE pET30a OYE 1 gene OYE 1 His-tag protein
pET21a(+) T-7 promoter, and AmpR
pDJB32 pET21a(+) OYE 2.6-GST fusion protein
OYE 2.6-GST fusion protein
pBS2 pDJB32 Y368 silent Deletion of NdeI restriction site
pET22b(+) T-7 promoter, and AmpR
pFBI pET22b(+) OYE 2.6 gene OYE 2.6 non-tagged protein
pYEX-41
pET26(+) T-7 promoter, and KanR
pDJB6 pET26(+)
pGEX
OYE 3-GST fusion protein OYE 3-GST fusion protein
pRP1 pDJB6 Y389 silent Deletion of NdeI restriction site
pRP2 pRP1 5’-CAAATG-3’ Deletion of NdeI restriction site
pRP3 pRP2 5’-CATATG-3’ Addition of NdeI restriction site
pET22b(+) T-7 promoter, and AmpR
pRP4 pET22(+) OYE 3 gene OYE 3 non-tagged protein
CASS-3 phoA promoter, and AmpR
pGF23 CASS-3 mALAS gene mALAS-GST fusion protein
pRP5 pGF23 L161 silent Deletion of KpnI restriction site
233
APPENDIX C PLASMID SEQUENCES
Sequence of pET3b-OYE1
1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT
51 CACAGTTAAA TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT
101 GCGCTCATCG TCATCCTCGG CACCGTCACC CTGGATGCTG TAGGCATAGG
151 CTTGGTTATG CCGGTACTGC CGGGCCTCTT GCGGGATATC GTCCATTCCG
201 ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA TGCGTTGATG
251 CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA
351 TCATGGCGAC CACACCCGTC CTGTGGATAT CCGGATATAG TTCCTCCTTT
401 CAGCAAAAAA CCCCTCAAGA CCCGTTTAGA GGCCCCAAGG GGTTATGCTA
451 GTTATTGCTC AGCGGTGGCA GCAGCCAACT CAGCTTCCTT TCGGGCTTTG
501 TTAGCAGCCG GATCCCGACC CATTTGCTGT CCACCAGTCA TGCTAGCCAT
551 ATGCCTTTGT TAGATCAGGA ACGCCATATC TGGCAAATAT GAATACTGCT
601 CGAAGTGGTA AAAACATTCA AAGTACGCTC TCTTAAAAGT GATACAAAGG
651 TGAGCTATGA AACTGGCATT GCTTCAACGT GATAGCTGAG CCCATATTCT
701 TTCATATATG ATTTTCACGT CTGATTTTTT AATAAAAGAT GCAGAGTGAA
751 AACATGCTCC TGCCCTACTG TGTGCATATT CAGCGCTCGT CAAAAAACCT
801 GTTGATATTT ATTTTCATTA AAATTGTTTA TCCTAAATAT ATCTAGTCGT
851 TAATTATATA AGGTAAACGG GATTGTGGGG GTGGGGAAGG TAGAACATCG
901 AACTATGGAA TATTCAATTA CTTTTTGTCC CAGCCTAATT TGAGAGCTTC
951 TTCATAGGTG GGGTAGTCAA TATAACCATG AGCAGACATC TGGTAGAAAG
1001 TATCTCTGTC ATATTTGTTC AGAGGTAGAC CTTTTTCCAA ACGATCAACC
1051 AAATCCGGGT TAGAAATGAA GAATCTACCG TAACCGATCA AGGTTCTCTT
1101 GTCCTTAACT TCTTCTCTAA CGACTTCTGG GTGGAGAGCA AAATTACCAG
1151 CTCTAATGAC TGGGCCCTTC CAGATGGAGT AAACAAAATC GTTGCTACCT
1201 CCTTCGTATT CACCCTCCCC TTCAGTCAAG AATGGGTTAG TTACACGAGG
1251 TTCAACCAAA TGAACAAAAG CTAAACGTTT TCCGGCTTTA GCTCTCTTTT
1301 CTAATTCACC AGCAACGTAA GCATATTGGG CAACAATGCC GGTCTCGGCA
1351 CCACCAGACA TACTGTTGAA AACACCGTAT GGGGACAATC TCAAACCAAC
1401 TTTTTCATGA CCAATGGCTT CGACAAGAGC ATCAACAACT TCCAAGGTGA
1451 AACGAGCTCT GTTTTCAATA GATCCACCAT ATTCATCGGT TCTAGTATTG
1501 GAATGAGGGT CCAAGAACTG GTTTAACAAG TAACCGTTAG CACTGTGAAT
1551 TTCAACACCA TCGGCACCAG CAGCAATAGA GTTCTTGGCA GCCTGGACGT
1601 ATTCCTTAAT GTATTGCTTG ATTTCGTCCT TGGTTAGGCT GTGTTGTGGG
1651 TTGTTGGCCT TCTTGGCCTT AGCTTCTTGC TCGGCATCCA TGAAAACGTT
1701 GTCAGAAGCT GAATCGTAAC GCAAACCATC TCTGGCAAGA TTGTCTGGGA
1751 AAGCAGCCCA ACCCAAAACC CATAACTGAA CCCAAACGAA CGATTTCTTT
1801 TCATGAATAG CGTTGAAGAT TTTGGTCCAT TCCACCATTT GTTCTTCCGA
1851 CCAAACACCT GGAGCGTTAT CGTAACCGCC GGCTTGTGGG GATATGAAGG
1901 CACCTTCAGT GATAATCATG GTACCAGGTC TTTGAGCACG TTGGGTGTAG
1951 TATTCGACTG CCCAGTCCCT GTTTGGGATA TTACCAGGGT GAAGAGCTCT
2001 CATTCTGGTC AATGGAGGAA TGACAGCACG GTGCAAAAGT TCATTGTTCC
2051 CGATCTTGAT TGGTTTGAAT AGGTTGGTGT CACCTAAAGC TTGTGGCTTA
2101 AAATCTTTTA CAAATGACAT ATGTATATCT CCTTCTTAAA GTTAAACAAA
2151 ATTATTTCTA GAGGGAAACC GTTGTGGTCT CCCTATAGTG AGTCGTATTA
2201 ATTTCGCGGG ATCGAGATCT CGATCCTCTA CGCCGGACGC ATCGTGGCCG
2251 GCATCACCGG CGCCACAGGT GCGGTTGCTG GCGCCTATAT CGCCGACATC
234
2301 ACCGATGGGG AAGATCGGGC TCGCCACTTC GGGCTCATGA GCGCTTGTTT
2351 CGGCGTGGGT ATGGTGGCAG GCCCCGTGGC CGGGGGACTG TTGGGCGCCA
2401 TCTCCTTGCA TGCACCATTC CTTGCGGCGG CGGTGCTCAA CGGCCTCAAC
2451 CTACTACTGG GCTGCTTCCT AATGCAGGAG TCGCATAAGG GAGAGCGTCG
2501 ACCGATGCCC TTGAGAGCCT TCAACCCAGT CAGCTCCTTC CGGTGGGCGC
2551 GGGGCATGAC TATCGTCGCC GCACTTATGA CTGTCTTCTT TATCATGCAA
2601 CTCGTAGGAC AGGTGCCGGC AGCGCTCTGG GTCATTTTCG GCGAGGACCG
2651 CTTTCGCTGG AGCGCGACGA TGATCGGCCT GTCGCTTGCG GTATTCGGAA
2701 TCTTGCACGC CCTCGCTCAA GCCTTCGTCA CTGGTCCCGC CACCAAACGT
2751 TTCGGCGAGA AGCAGGCCAT TATCGCCGGC ATGGCGGCCG ACGCGCTGGG
2801 CTACGTCTTG CTGGCGTTCG CGACGCGAGG CTGGATGGCC TTCCCCATTA
2851 TGATTCTTCT CGCTTCCGGC GGCATCGGGA TGCCCGCGTT GCAGGCCATG
2901 CTGTCCAGGC AGGTAGATGA CGACCATCAG GGACAGCTTC AAGGATCGCT
2951 CGCGGCTCTT ACCAGCCTAA CTTCGATCAC TGGACCGCTG ATCGTCACGG
3001 CGATTTATGC CGCCTCGGCG AGCACATGGA ACGGGTTGGC ATGGATTGTA
3051 GGCGCCGCCC TATACCTTGT CTGCCTCCCC GCGTTGCGTC GCGGTGCATG
3101 GAGCCGGGCC ACCTCGACCT GAATGGAAGC CGGCGGCACC TCGCTAACGG
3151 ATTCACCACT CCAAGAATTG GAGCCAATCA ATTCTTGCGG AGAACTGTGA
3201 ATGCGCAAAC CAACCCTTGG CAGAACATAT CCATCGCGTC CGCCATCTCC
3251 AGCAGCCGCA CGCGGCGCAT CTCGGGCAGC GTTGGGTCCT GGCCACGGGT
3301 GCGCATGATC GTGCTCCTGT CGTTGAGGAC CCGGCTAGGC TGGCGGGGTT
3351 GCCTTACTGG TTAGCAGAAT GAATCACCGA TACGCGAGCG AACGTGAAGC
3401 GACTGCTGCT GCAAAACGTC TGCGACCTGA GCAACAACAT GAATGGTCTT
3451 CGGTTTCCGT GTTTCGTAAA GTCTGGAAAC GCGGAAGTCA GCGCCCTGCA
3501 CCATTATGTT CCGGATCTGC ATCGCAGGAT GCTGCTGGCT ACCCTGTGGA
3551 ACACCTACAT CTGTATTAAC GAAGCGCTGG CATTGACCCT GAGTGATTTT
3601 TCTCTGGTCC CGCCGCATCC ATACCGCCAG TTGTTTACCC TCACAACGTT
3651 CCAGTAACCG GGCATGTTCA TCATCAGTAA CCCGTATCGT GAGCATCCTC
3701 TCTCGTTTCA TCGGTATCAT TACCCCCATG AACAGAAATC CCCCTTACAC
3751 GGAGGCATCA GTGACCAAAC AGGAAAAAAC CGCCCTTAAC ATGGCCCGCT
3801 TTATCAGAAG CCAGACATTA ACGCTTCTGG AGAAACTCAA CGAGCTGGAC
3851 GCGGATGAAC AGGCAGACAT CTGTGAATCG CTTCACGACC ACGCTGATGA
3901 GCTTTACCGC AGCTGCCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT
3951 GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC
4001 GGGAGCAGAC AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG
4051 GGGCGCAGCC ATGACCCAGT CACGTAGCGA TAGCGGAGTG TATACTGGCT
4101 TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATATGCG
4151 GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCT
4201 CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT TCGGCTGCGG
4251 CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC
4301 AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA
4351 GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC
4401 CCTGACGAGC ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC
4451 GACAGGACTA TAAAGATACC AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC
4501 GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC CGCCTTTCTC
4551 CCTTCGGGAA GCGTGGCGCT TTCTCATAGC TCACGCTGTA GGTATCTCAG
4601 TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG
4651 TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC
4701 CCGGTAAGAC ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT
4751 TAGCAGAGCG AGGTATGTAG GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC
4801 CTAACTACGG CTACACTAGA AGGACAGTAT TTGGTATCTG CGCTCTGCTG
4851 AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT CCGGCAAACA
235
4901 AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC
4951 GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT
5001 GACGCTCAGT GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT
5051 ATCAAAAAGG ATCTTCACCT AGATCCTTTT AAATTAAAAA TGAAGTTTTA
5101 AATCAATCTA AAGTATATAT GAGTAAACTT GGTCTGACAG TTACCAATGC
5151 TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC GTTCATCCAT
5201 AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC
5251 CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT
5301 CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG
5351 TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG
5401 AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC
5451 ATTGCTGCAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA TGGCTTCATT
5501 CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT
5551 GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG
5601 TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT
5651 TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA
5701 CCAAGTCATT CTGAGAATAG TGTATGCGGC GACCGAGTTG CTCTTGCCCG
5751 GCGTCAACAC GGGATAATAC CGCGCCACAT AGCAGAACTT TAAAAGTGCT
5801 CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC
5851 TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA
5901 GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA
5951 AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA
6001 TACTCTTCCT TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC
6051 ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC AAATAGGGGT
6101 TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTCTAA GAAACCATTA
6151 TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT
6201 CTTCAAGAA
Sequence of pBS2
1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG
51 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT
101 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG
151 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC
201 GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG
251 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT
301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT
351 CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG
401 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT
451 ATTAACGTTT ACAATTTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA
501 CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG
551 AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT
601 GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT
651 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT
701 GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG
751 CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA
801 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC
851 GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT
901 TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA
951 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC
1001 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA
236
1051 CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA
1101 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGCAGCAATG
1151 GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC
1201 CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC
1251 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA
1301 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG
1351 TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA
1401 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG
1451 CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT
1501 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA
1551 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA
1601 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG
1651 CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT
1701 GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC
1751 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG
1801 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA
1851 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG
1901 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC
1951 GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC
2001 TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG
2051 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG
2101 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG
2151 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG
2201 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT
2251 GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG
2301 ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC
2351 CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA
2401 GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC
2451 GCATATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA
2501 GCCAGTATAC ACTCCGCTAT CGCTACGTGA CTGGGTCATG GCTGCGCCCC
2551 GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG
2601 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA
2651 GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCTG CGGTAAAGCT
2701 CATCAGCGTG GTCGTGAAGC GATTCACAGA TGTCTGCCTG TTCATCCGCG
2751 TCCAGCTCGT TGAGTTTCTC CAGAAGCGTT AATGTCTGGC TTCTGATAAA
2801 GCGGGCCATG TTAAGGGCGG TTTTTTCCTG TTTGGTCACT GATGCCTCCG
2851 TGTAAGGGGG ATTTCTGTTC ATGGGGGTAA TGATACCGAT GAAACGAGAG
2901 AGGATGCTCA CGATACGGGT TACTGATGAT GAACATGCCC GGTTACTGGA
2951 ACGTTGTGAG GGTAAACAAC TGGCGGTATG GATGCGGCGG GACCAGAGAA
3001 AAATCACTCA GGGTCAATGC CAGCGCTTCG TTAATACAGA TGTAGGTGTT
3051 CCACAGGGTA GCCAGCAGCA TCCTGCGATG CAGATCCGGA ACATAATGGT
3101 GCAGGGCGCT GACTTCCGCG TTTCCAGACT TTACGAAACA CGGAAACCGA
3151 AGACCATTCA TGTTGTTGCT CAGGTCGCAG ACGTTTTGCA GCAGCAGTCG
3201 CTTCACGTTC GCTCGCGTAT CGGTGATTCA TTCTGCTAAC CAGTAAGGCA
3251 ACCCCGCCAG CCTAGCCGGG TCCTCAACGA CAGGAGCACG ATCATGCGCA
3301 CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT
3351 TTGGTGGCGG GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC
3401 GAATACCGCA AGCGACAGGC CGATCATCGT CGCGCTCCAG CGAAAGCGGT
3451 CCTCGCCGAA AATGACCCAG AGCGCTGCCG GCACCTGTCC TACGAGTTGC
3501 ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA TGCCCCGCGC
3551 CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG
3601 ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA
237
3651 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT
3701 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC CAGGGTGGTT
3751 TTTCTTTTCA CCAGTGAGAC GGGCAACAGC TGATTGCCCT TCACCGCCTG
3801 GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC CCCAGCAGGC
3851 GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT
3901 TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC
3951 GGACTCGGTA ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA
4001 CCAGCATCGC AGTGGGAACG ATGCCCTCAT TCAGCATTTG CATGGTTTGT
4051 TGAAAACCGG ACATGGCACT CCAGTCGCCT TCCCGTTCCG CTATCGGCTG
4101 AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA CGCAGACGCG
4151 CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC
4201 AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA
4251 AATAATACTG TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG
4301 GAACATTAGT GCAGGCAGCT TCCACAGCAA TGGCATCCTG GTCATCCAGC
4351 GGATAGTTAA TGATCAGCCC ACTGACGCGT TGCGCGAGAA GATTGTGCAC
4401 CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC GACACCACCA
4451 CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC
4501 GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA
4551 CTGTTTGCCC GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT
4601 CCGCCATCGC CGCTTCCACT TTTTCCCGCG TTTTCGCAGA AACGTGGCTG
4651 GCCTGGTTCA CCACGCGGGA AACGGTCTGA TAAGAGACAC CGGCATACTC
4701 TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC CTGAATTGAC
4751 TCTCTTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG
4801 ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA
4851 AGCAGCCCAG TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT
4901 GGTGCATGCA AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC
4951 CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC
5001 GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC AACCGCACCT
5051 GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCGAGAT
5101 CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA
5151 TAACAATTCC CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT
5201 ACATAATGAC CAAGTTACCT ATACTAGGTT ATTGGAAAAT TAAGGGCCTT
5251 GTGCAACCCA CTCGACTTCT TTTGGAATAT CTTGAAGAAA AATATGAAGA
5301 GCATTTGTAT GAGCGCGATG AAGGTGATAA ATGGCGAAAC AAAAAGTTTG
5351 AATTGGGTTT GGAGTTTCCC AATCTTCCTT ATTATATTGA TGGTGATGTT
5401 AAATTAACAC AGTCTATGGC CATCATACGT TATATAGCTG ACAAGCACAA
5451 CATGTTGGGT GGTTGTCCAA AAGAGCGTGC AGAGATTTCA ATGCTTGAAG
5501 GAGCGGTTTT GGATATTAGA TACGGTGTTT CGAGAATTGC ATATAGTAAA
5551 GACTTTGAAA CTCTCAAAGT TGATTTTCTT AGCAAGCTAC CTGAAATGCT
5601 GAAAATGTTC GAAGATCGTT TATGTCATAA AACATATTTA AATGGTGATC
5651 ATGTAACCCA TCCTGACTTC ATGTTGTATG ACGCTCTTGA TGTTGTTTTA
5701 TACATGGACC CAATGTGCCT GGATGCGTTC CCAAAATTAG TTTGTTTTAA
5751 AAAACGTATT GAAGCTATCC CACAAATTGA TAAGTACTTG AAATCCAGCA
5801 AGTATATAGC ATGGCCTTTG CAGGGCTGGC AAGCCACGTT TGGTGGTGGC
5851 GACCATCCTC CAAAATCGGA TCATCTGGTT CCGCGTCATA TGCCCATGTC
5901 TTCAGTCAAA ATTTCTCCAT TGAAGGATTC TGAAGCATTC CAGTCTATCA
5951 AAGTTGGTAA CAACACTCTT CAAACCAAGA TTGTCTATCC ACCAACTACT
6001 AGATTTAGAG CTTTAGAAGA CCACACTCCT TCTGATTTGC AATTGCAGTA
6051 CTATGGCGAC AGATCCACTT TCCCAGGTAC TTTGCTTATC ACTGAAGCTA
6101 CTTTTGTCTC TCCTCAAGCC TCTGGTTATG AAGGTGCTGC TCCAGGTATT
6151 TGGACTGACA AGCACGCTAA AGCATGGAAG GTTATTACTG ATAAAGTTCA
6201 TGCCAACGGT TCTTTCGTTT CAACCCAGTT GATTTTTTTG GGAAGGGTTG
238
6251 CAGATCCAGC TGTTATGAAG ACCCGTGGGT TGAATCCAGT TTCTGCCTCT
6301 GCTACTTATG AAAGTGATGC CGCTAAAGAA GCTGCCGAAG CAGTTGGTAA
6351 CCCTGTTAGA GCTTTGACTA CCCAAGAAGT CAAGGATCTT GTTTACGAGG
6401 CTTACACCAA CGCTGCTCAG AAGGCCATGG ATGCTGGTTT CGACTATATT
6451 GAACTCCATG CTGCTCATGG CTACCTTTTA GATCAATTTT TGCAACCATG
6501 CACCAATCAA AGAACTGATG AATACGGTGG ATCCATTGAG AACAGAGCCA
6551 GGTTAATTCT TGAGTTGATT GACCATTTGT CTACCATTGT CGGTGCTGAC
6601 AAGATTGGTA TCAGAATCTC TCCATGGGCT ACTTTCCAAA ACATGAAGGC
6651 TCACAAGGAC ACTGTTCACC CATTGACTAC TTTCTCTTAC TTGGTCCACG
6701 AATTGCAACA GAGAGCTGAC AAGGGTCAAG GTATTGCCTA CATTTCTGTC
6751 GTTGAGCCTC GTGTAAGTGG TAACGTCGAC GTCTCTGAAG AAGACCAAGC
6801 TGGTGACAAC GAATTTGTCT CCAAGATCTG GAAGGGTGTT ATCTTGAAGG
6851 CAGGTAACTA CTCCTACGAT GCTCCAGAGT TCAAGACATT GAAGGAAGAT
6901 ATCGCTGACA AGCGTACATT AGTTGGCTTC TCCAGATACT TCACCTCGAA
6951 TCCTAACTTG GTTTGGAAAT TGCGTGATGG AATTGACTTG GTGCCATACG
7001 ACAGAAACAC GTTCTACAGT GACAATAACT ATGGTTACAA TACCTTTTCT
7051 ATGGATTCCG AAGAGGTTGA TAAAGAATTA GAAATCAAGA GAGTTCCTTC
7101 GGCCATTGAA GCTTTGTGAT GCGGCCGCAC TCGAGCACCA CCACCACCAC
7151 CACTGAGATC CGGCTGCTAA CAAAGCCCGA AAGGAAGCTG AGTTGGCTGC
7201 TGCCACCGCT GAGCAATAAC TAGCATAACC CCTTGGGGCC TCTAAACGGG
7251 TCTTGAGGGG TTTTTTGCTG AAAGGAGGAA CTATATCCGG AT
Sequence of pFB1
1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG
51 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT
101 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG
151 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC
201 GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG
251 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT
301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT
351 CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG
401 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT
451 ATTAACGTTT ACAATTTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA
501 CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG
551 AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT
601 GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT
651 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT
701 GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG
751 CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA
801 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC
851 GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT
901 TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA
951 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC
1001 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA
1051 CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA
1101 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGCAGCAATG
1151 GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC
1201 CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC
1251 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA
1301 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG
239
1351 TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA
1401 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG
1451 CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT
1501 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA
1551 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA
1601 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG
1651 CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT
1701 GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC
1751 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG
1801 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA
1851 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG
1901 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC
1951 GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC
2001 TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG
2051 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG
2101 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG
2151 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG
2201 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT
2251 GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG
2301 ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC
2351 CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA
2401 GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC
2451 GCATATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA
2501 GCCAGTATAC ACTCCGCTAT CGCTACGTGA CTGGGTCATG GCTGCGCCCC
2551 GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG
2601 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA
2651 GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCTG CGGTAAAGCT
2701 CATCAGCGTG GTCGTGAAGC GATTCACAGA TGTCTGCCTG TTCATCCGCG
2751 TCCAGCTCGT TGAGTTTCTC CAGAAGCGTT AATGTCTGGC TTCTGATAAA
2801 GCGGGCCATG TTAAGGGCGG TTTTTTCCTG TTTGGTCACT GATGCCTCCG
2851 TGTAAGGGGG ATTTCTGTTC ATGGGGGTAA TGATACCGAT GAAACGAGAG
2901 AGGATGCTCA CGATACGGGT TACTGATGAT GAACATGCCC GGTTACTGGA
2951 ACGTTGTGAG GGTAAACAAC TGGCGGTATG GATGCGGCGG GACCAGAGAA
3001 AAATCACTCA GGGTCAATGC CAGCGCTTCG TTAATACAGA TGTAGGTGTT
3051 CCACAGGGTA GCCAGCAGCA TCCTGCGATG CAGATCCGGA ACATAATGGT
3101 GCAGGGCGCT GACTTCCGCG TTTCCAGACT TTACGAAACA CGGAAACCGA
3151 AGACCATTCA TGTTGTTGCT CAGGTCGCAG ACGTTTTGCA GCAGCAGTCG
3201 CTTCACGTTC GCTCGCGTAT CGGTGATTCA TTCTGCTAAC CAGTAAGGCA
3251 ACCCCGCCAG CCTAGCCGGG TCCTCAACGA CAGGAGCACG ATCATGCGCA
3301 CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT
3351 TTGGTGGCGG GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC
3401 GAATACCGCA AGCGACAGGC CGATCATCGT CGCGCTCCAG CGAAAGCGGT
3451 CCTCGCCGAA AATGACCCAG AGCGCTGCCG GCACCTGTCC TACGAGTTGC
3501 ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA TGCCCCGCGC
3551 CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG
3601 ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA
3651 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT
3701 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC CAGGGTGGTT
3751 TTTCTTTTCA CCAGTGAGAC GGGCAACAGC TGATTGCCCT TCACCGCCTG
3801 GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC CCCAGCAGGC
3851 GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT
3901 TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC
240
3951 GGACTCGGTA ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA
4001 CCAGCATCGC AGTGGGAACG ATGCCCTCAT TCAGCATTTG CATGGTTTGT
4051 TGAAAACCGG ACATGGCACT CCAGTCGCCT TCCCGTTCCG CTATCGGCTG
4101 AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA CGCAGACGCG
4151 CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC
4201 AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA
4251 AATAATACTG TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG
4301 GAACATTAGT GCAGGCAGCT TCCACAGCAA TGGCATCCTG GTCATCCAGC
4351 GGATAGTTAA TGATCAGCCC ACTGACGCGT TGCGCGAGAA GATTGTGCAC
4401 CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC GACACCACCA
4451 CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC
4501 GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA
4551 CTGTTTGCCC GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT
4601 CCGCCATCGC CGCTTCCACT TTTTCCCGCG TTTTCGCAGA AACGTGGCTG
4651 GCCTGGTTCA CCACGCGGGA AACGGTCTGA TAAGAGACAC CGGCATACTC
4701 TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC CTGAATTGAC
4751 TCTCTTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG
4801 ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA
4851 AGCAGCCCAG TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT
4901 GGTGCATGCA AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC
4951 CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC
5001 GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC AACCGCACCT
5051 GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCGAGAT
5101 CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA
5151 TAACAATTCC CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT
5201 ACATATGCCC ATGTCTTCAG TCAAAATTTC TCCATTGAAG GATTCTGAAG
5251 CATTCCAGTC TATCAAAGTT GGTAACAACA CTCTTCAAAC CAAGATTGTC
5301 TATCCACCAA CTACTAGATT TAGAGCTTTA GAAGACCACA CTCCTTCTGA
5351 TTTGCAATTG CAGTACTATG GCGACAGATC CACTTTCCCA GGTACTTTGC
5401 TTATCACTGA AGCTACTTTT GTCTCTCCTC AAGCCTCTGG TTATGAAGGT
5451 GCTGCTCCAG GTATTTGGAC TGACAAGCAC GCTAAAGCAT GGAAGGTTAT
5501 TACTGATAAA GTTCATGCCA ACGGTTCTTT CGTTTCAACC CAGTTGATTT
5551 TTTTGGGAAG GGTTGCAGAT CCAGCTGTTA TGAAGACCCG TGGGTTGAAT
5601 CCAGTTTCTG CCTCTGCTAC TTATGAAAGT GATGCCGCTA AAGAAGCTGC
5651 CGAAGCAGTT GGTAACCCTG TTAGAGCTTT GACTACCCAA GAAGTCAAGG
5701 ATCTTGTTTA CGAGGCTTAC ACCAACGCTG CTCAGAAGGC CATGGATGCT
5751 GGTTTCGACT ATATTGAACT CCATGCTGCT CATGGCTACC TTTTAGATCA
5801 ATTTTTGCAA CCATGCACCA ATCAAAGAAC TGATGAATAC GGTGGATCCA
5851 TTGAGAACAG AGCCAGGTTA ATTCTTGAGT TGATTGACCA TTTGTCTACC
5901 ATTGTCGGTG CTGACAAGAT TGGTATCAGA ATCTCTCCAT GGGCTACTTT
5951 CCAAAACATG AAGGCTCACA AGGACACTGT TCACCCATTG ACTACTTTCT
6001 CTTACTTGGT CCACGAATTG CAACAGAGAG CTGACAAGGG TCAAGGTATT
6051 GCCTACATTT CTGTCGTTGA GCCTCGTGTA AGTGGTAACG TCGACGTCTC
6101 TGAAGAAGAC CAAGCTGGTG ACAACGAATT TGTCTCCAAG ATCTGGAAGG
6151 GTGTTATCTT GAAGGCAGGT AACTACTCCT ACGATGCTCC AGAGTTCAAG
6201 ACATTGAAGG AAGATATCGC TGACAAGCGT ACATTAGTTG GCTTCTCCAG
6251 ATACTTCACC TCGAATCCTA ACTTGGTTTG GAAATTGCGT GATGGAATTG
6301 ACTTGGTGCC ATACGACAGA AACACGTTCT ACAGTGACAA TAACTATGGT
6351 TACAATACCT TTTCTATGGA TTCCGAAGAG GTTGATAAAG AATTAGAAAT
6401 CAAGAGAGTT CCTTCGGCCA TTGAAGCTTT GTGATGCGGC CGCACTCGAG
6451 CACCACCACC ACCACCACTG AGATCCGGCT GCTAACAAAG CCCGAAAGGA
6501 AGCTGAGTTG GCTGCTGCCA CCGCTGAGCA ATAACTAGCA TAACCCCTTG
241
6551 GGGCCTCTAA ACGGGTCTTG AGGGGTTTTT TGCTGAAAGG AGGAACTATA
6601 TCCGGAT
Sequence of pRP4
1 TGGCGAATGG GACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG
51 TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT
101 CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG
151 TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC
201 GGCACCTCGA CCCCAAAAAA CTTGATTAGG GTGATGGTTC ACGTAGTGGG
251 CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT
301 CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT
351 CGGTCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG
401 TTAAAAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT
451 ATTAACGTTT ACAATTTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA
501 CCCCTATTTG TTTATTTTTC TAAATACATT CAAATATGTA TCCGCTCATG
551 AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA GGAAGAGTAT
601 GAGTATTCAA CATTTCCGTG TCGCCCTTAT TCCCTTTTTT GCGGCATTTT
651 GCCTTCCTGT TTTTGCTCAC CCAGAAACGC TGGTGAAAGT AAAAGATGCT
701 GAAGATCAGT TGGGTGCACG AGTGGGTTAC ATCGAACTGG ATCTCAACAG
751 CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGTTTT CCAATGATGA
801 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC
851 GGGCAAGAGC AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT
901 TGAGTACTCA CCAGTCACAG AAAAGCATCT TACGGATGGC ATGACAGTAA
951 GAGAATTATG CAGTGCTGCC ATAACCATGA GTGATAACAC TGCGGCCAAC
1001 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG CTTTTTTGCA
1051 CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA
1101 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGCAGCAATG
1151 GCAACAACGT TGCGCAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC
1201 CCGGCAACAA TTAATAGACT GGATGGAGGC GGATAAAGTT GCAGGACCAC
1251 TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT TTATTGCTGA TAAATCTGGA
1301 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG GGCCAGATGG
1351 TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA
1401 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG
1451 CATTGGTAAC TGTCAGACCA AGTTTACTCA TATATACTTT AGATTGATTT
1501 AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC CTTTTTGATA
1551 ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA
1601 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG
1651 CGTAATCTGC TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT
1701 GTTTGCCGGA TCAAGAGCTA CCAACTCTTT TTCCGAAGGT AACTGGCTTC
1751 AGCAGAGCGC AGATACCAAA TACTGTCCTT CTAGTGTAGC CGTAGTTAGG
1801 CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC GCTCTGCTAA
1851 TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG
1901 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC
1951 GGGGGGTTCG TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC
2001 TGAGATACCT ACAGCGTGAG CTATGAGAAA GCGCCACGCT TCCCGAAGGG
2051 AGAAAGGCGG ACAGGTATCC GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG
2101 CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT AGTCCTGTCG
2151 GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG
2201 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT
2251 GGCCTTTTGC TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG
242
2301 ATTCTGTGGA TAACCGTATT ACCGCCTTTG AGTGAGCTGA TACCGCTCGC
2351 CGCAGCCGAA CGACCGAGCG CAGCGAGTCA GTGAGCGAGG AAGCGGAAGA
2401 GCGCCTGATG CGGTATTTTC TCCTTACGCA TCTGTGCGGT ATTTCACACC
2451 GCATATATGG TGCACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAA
2501 GCCAGTATAC ACTCCGCTAT CGCTACGTGA CTGGGTCATG GCTGCGCCCC
2551 GACACCCGCC AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG
2601 GCATCCGCTT ACAGACAAGC TGTGACCGTC TCCGGGAGCT GCATGTGTCA
2651 GAGGTTTTCA CCGTCATCAC CGAAACGCGC GAGGCAGCTG CGGTAAAGCT
2701 CATCAGCGTG GTCGTGAAGC GATTCACAGA TGTCTGCCTG TTCATCCGCG
2751 TCCAGCTCGT TGAGTTTCTC CAGAAGCGTT AATGTCTGGC TTCTGATAAA
2801 GCGGGCCATG TTAAGGGCGG TTTTTTCCTG TTTGGTCACT GATGCCTCCG
2851 TGTAAGGGGG ATTTCTGTTC ATGGGGGTAA TGATACCGAT GAAACGAGAG
2901 AGGATGCTCA CGATACGGGT TACTGATGAT GAACATGCCC GGTTACTGGA
2951 ACGTTGTGAG GGTAAACAAC TGGCGGTATG GATGCGGCGG GACCAGAGAA
3001 AAATCACTCA GGGTCAATGC CAGCGCTTCG TTAATACAGA TGTAGGTGTT
3051 CCACAGGGTA GCCAGCAGCA TCCTGCGATG CAGATCCGGA ACATAATGGT
3101 GCAGGGCGCT GACTTCCGCG TTTCCAGACT TTACGAAACA CGGAAACCGA
3151 AGACCATTCA TGTTGTTGCT CAGGTCGCAG ACGTTTTGCA GCAGCAGTCG
3201 CTTCACGTTC GCTCGCGTAT CGGTGATTCA TTCTGCTAAC CAGTAAGGCA
3251 ACCCCGCCAG CCTAGCCGGG TCCTCAACGA CAGGAGCACG ATCATGCGCA
3301 CCCGTGGGGC CGCCATGCCG GCGATAATGG CCTGCTTCTC GCCGAAACGT
3351 TTGGTGGCGG GACCAGTGAC GAAGGCTTGA GCGAGGGCGT GCAAGATTCC
3401 GAATACCGCA AGCGACAGGC CGATCATCGT CGCGCTCCAG CGAAAGCGGT
3451 CCTCGCCGAA AATGACCCAG AGCGCTGCCG GCACCTGTCC TACGAGTTGC
3501 ATGATAAAGA AGACAGTCAT AAGTGCGGCG ACGATAGTCA TGCCCCGCGC
3551 CCACCGGAAG GAGCTGACTG GGTTGAAGGC TCTCAAGGGC ATCGGTCGAG
3601 ATCCCGGTGC CTAATGAGTG AGCTAACTTA CATTAATTGC GTTGCGCTCA
3651 CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT
3701 CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC CAGGGTGGTT
3751 TTTCTTTTCA CCAGTGAGAC GGGCAACAGC TGATTGCCCT TCACCGCCTG
3801 GCCCTGAGAG AGTTGCAGCA AGCGGTCCAC GCTGGTTTGC CCCAGCAGGC
3851 GAAAATCCTG TTTGATGGTG GTTAACGGCG GGATATAACA TGAGCTGTCT
3901 TCGGTATCGT CGTATCCCAC TACCGAGATA TCCGCACCAA CGCGCAGCCC
3951 GGACTCGGTA ATGGCGCGCA TTGCGCCCAG CGCCATCTGA TCGTTGGCAA
4001 CCAGCATCGC AGTGGGAACG ATGCCCTCAT TCAGCATTTG CATGGTTTGT
4051 TGAAAACCGG ACATGGCACT CCAGTCGCCT TCCCGTTCCG CTATCGGCTG
4101 AATTTGATTG CGAGTGAGAT ATTTATGCCA GCCAGCCAGA CGCAGACGCG
4151 CCGAGACAGA ACTTAATGGG CCCGCTAACA GCGCGATTTG CTGGTGACCC
4201 AATGCGACCA GATGCTCCAC GCCCAGTCGC GTACCGTCTT CATGGGAGAA
4251 AATAATACTG TTGATGGGTG TCTGGTCAGA GACATCAAGA AATAACGCCG
4301 GAACATTAGT GCAGGCAGCT TCCACAGCAA TGGCATCCTG GTCATCCAGC
4351 GGATAGTTAA TGATCAGCCC ACTGACGCGT TGCGCGAGAA GATTGTGCAC
4401 CGCCGCTTTA CAGGCTTCGA CGCCGCTTCG TTCTACCATC GACACCACCA
4451 CGCTGGCACC CAGTTGATCG GCGCGAGATT TAATCGCCGC GACAATTTGC
4501 GACGGCGCGT GCAGGGCCAG ACTGGAGGTG GCAACGCCAA TCAGCAACGA
4551 CTGTTTGCCC GCCAGTTGTT GTGCCACGCG GTTGGGAATG TAATTCAGCT
4601 CCGCCATCGC CGCTTCCACT TTTTCCCGCG TTTTCGCAGA AACGTGGCTG
4651 GCCTGGTTCA CCACGCGGGA AACGGTCTGA TAAGAGACAC CGGCATACTC
4701 TGCGACATCG TATAACGTTA CTGGTTTCAC ATTCACCACC CTGAATTGAC
4751 TCTCTTCCGG GCGCTATCAT GCCATACCGC GAAAGGTTTT GCGCCATTCG
4801 ATGGTGTCCG GGATCTCGAC GCTCTCCCTT ATGCGACTCC TGCATTAGGA
4851 AGCAGCCCAG TAGTAGGTTG AGGCCGTTGA GCACCGCCGC CGCAAGGAAT
243
4901 GGTGCATGCA AGGAGATGGC GCCCAACAGT CCCCCGGCCA CGGGGCCTGC
4951 CACCATACCC ACGCCGAAAC AAGCGCTCAT GAGCCCGAAG TGGCGAGCCC
5001 GATCTTCCCC ATCGGTGATG TCGGCGATAT AGGCGCCAGC AACCGCACCT
5051 GTGGCGCCGG TGATGCCGGC CACGATGCGT CCGGCGTAGA GGATCGAGAT
5101 CTCGATCCCG CGAAATTAAT ACGACTCACT ATAGGGGAAT TGTGAGCGGA
5151 TAACAATTCC CCTCTAGAAA TAATTTTGTT TAACTTTAAG AAGGAGATAT
5201 ACATATGCCA TTTGTAAAAG GTTTTGAGCC GATCTCCCTA AGAGACACAA
5251 ACCTTTTTGA ACCAATTAAG ATTGGTAACA CTCAGCTTGC ACATCGTGCG
5301 GTTATGCCCC CATTGACCAG AATGAGGGCC ACTCACCCCG GAAATATTCC
5351 AAATAAGGAG TGGGCTGCTG TGTATTATGG TCAGCGTGCT CAAAGACCTG
5401 GTACCATGAT CATCACGGAA GGTACGTTTA TTTCCCCTCA AGCCGGCGGC
5451 TATGACAACG CCCCTGGGAT TTGGTCTGAT GAGCAGGTCG CTGAGTGGAA
5501 GAATATCTTT TTAGCCATCC ATGATTGTCA GTCGTTCGCG TGGGTACAAC
5551 TTTGGTCTTT AGGCTGGGCA TCCTTCCCAG ACGTATTGGC AAGAGACGGG
5601 TTACGCTATG ACTGTGCATC TGACAGAGTG TATATGAATG CTACGTTACA
5651 AGAAAAGGCC AAAGATGCGA ATAATCTCGA ACATAGTTTG ACTAAAGACG
5701 ACATTAAACA GTATATCAAG GATTACATCC ATGCGGCTAA GAATTCTATC
5751 GCGGCTGGCG CCGATGGTGT AGAAATTCAT AGCGCCAATG GGTACTTGTT
5801 GAATCAGTTC TTGGATCCAC ATTCTAATAA GAGGACCGAC GAATACGGCG
5851 GAACGATCGA AAACAGGGCC CGCTTTACAC TGGAGGTTGT CGATGCTCTT
5901 ATCGAAACTA TCGGTCCTGA ACGGGTGGGT TTGAGGTTGT CGCCGTACGG
5951 CACTTTTAAC AGTATGTCTG GGGGTGCTGA ACCAGGTATT ATCGCTCAAT
6001 ATTCGTATGT TTTGGGTGAA TTAGAGAAGA GGGCAAAGGC TGGTAAGCGT
6051 TTGGCCTTTG TGCACCTCGT TGAACCACGT GTCACGGACC CATCGTTGGT
6101 GGAGGGCGAA GGAGAATATT CCGAGGGTAC TAACGATTTT GCCTACTCTA
6151 TATGGAAGGG TCCAATCATC AGAGCTGGTA ATTACGCTCT TCATCCAGAA
6201 GTGGTTAGAG AACAAGTAAA GGATCCCAGA ACCTTGATAG GCTATGGTAG
6251 ATTCTTCATC TCTAACCCAG ATTTAGTCTA CCGTTTAGAA GAGGGCCTGC
6301 CATTGAACAA GTATGACAGA AGTACCTTCT ACACCATGTC CGCGGAAGGT
6351 TATACCGACT ACCCAACATA CGAAGAGGCA GTAGATTTAG GTTGGAACAA
6401 GAACTGATGC GGCCGCACTC GAGCACCACC ACCACCACCA CTGAGATCCG
6451 GCTGCTAACA AAGCCCGAAA GGAAGCTGAG TTGGCTGCTG CCACCGCTGA
6501 GCAATAACTA GCATAACCCC TTGGGGCCTC TAAACGGGTC TTGAGGGGTT
6551 TTTTGCTGAA AGGAGGAACT ATATCCGGAT
Sequence of pGF23
1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTGGAGA TTATCGTCAC
51 TGCAATGCTT CGCAATATGG CGCAAAATGA CCAACAGCGG TTGATTGATC
101 AGGTAGAGGG GGCGCTGTAC GAGGTAAAGC CCGATGCCAG CATTCCTGAC
151 GACGATACGG AGCTGCTGCG CGATTACGTA AAGAAGTTAT TGAAGCATCC
201 TCGTCAGTAA AAAGTTAATC TTTTCAACAG CTGTCATAAA GTTGTCACGG
251 CCGAGACTTA TAGTCGCTTT GTTTTTATTT TTTAATGTAT TTGTACATGG
301 AGAAAATAAA GTGAAACAGT CGACTGACAG GAAGAGCAAG ATTGTGCAGA
351 GGGCAGCTCC AGAAGTTCAA GAGGATGTCA AGACTTTCAA GACAGACCTG
401 CTGAGCACCA TGGATTCAAC CACCCGAAGC CATTCATTTC CTAGTTTCCA
451 GGAGCCAGAG CAGACTGAAG GGGCAGTTCC CCACCTGATT CAGAACAATA
501 TGACTGGAAG CCAGGCTTTC GGTTATGACC AATTTTTCAG AGACAAGATC
551 ATGGAGAAGA AACAGGACCA CACCTACCGT GTGTTCAAGA CTGTGAATCG
601 TTGGGCTAAT GCCTACCCCT TTGCCCAACA CTTCTCCGAG GCATCTATGG
651 CATCAAAGGA TGTTTCTGTT TGGTGTAGTA ATGACTATTT GGGCATAAGC
244
701 AGACACCCTC GTGTCTTGCA GGCCATAGAG GAGACCCTGA AGAATCATGG
751 AGCTGGAGCT GGGGGCACTC GCAATATCTC AGGTACCAGC AAGTTTCATG
801 TGGAGCTTGA ACAGGAGCTG GCTGAACTAC ACCAGAAAGA CTCAGCTCTG
851 CTCTTCTCCT CCTGTTTTGT GGCCAATGAT TCTACTCTCT TTACACTGGC
901 CAAGCTTCTG CCAGGGTGTG AGATCTACTC AGATGCAGGC AATCATGCCT
951 CCATGATCCA AGGCATTCGC AACAGTGGTG CAGCCAAGTT TGTCTTCAGA
1001 CACAATGACC CAGGCCACCT GAAGAAACTT CTCGAGAAGT CTGATCCCAA
1051 GACACCAAAA ATTGTGGCTT TTGAGACTGT TCATTCCATG GATGGTGCCA
1101 TCTGTCCTCT GGAGGAATTG TGTGATGTGG CCCACCAGTA TGGAGCCCTG
1151 ACCTTCGTAG ATGAAGTCCA TGCTGTAGGA CTGTATGGAG CCCGGGGTGC
1201 AGGTATCGGG GAGCGTGATG GAATTATGCA CAAGCTTGAC ATCATCTCTG
1251 GAACTCTTGG CAAGGCCTTT GGTTGCGTCG GTGGCTATAT AGCCAGCACT
1301 CGGGACTTGG TGGACATGGT GCGCTCCTAC GCTGCAGGCT TCATCTTTAC
1351 CACTTCACTG CCTCCCATGA TGCTCTCTGG GGCTCTAGAA TCTGTGCGCC
1401 TACTCAAGGG AGAGGAGGGT CAAGCCCTGA GGCGGGCACA CCAGCGCAAT
1451 GTCAAACACA TGCGCCAGCT GCTAATGGAC AGGGGCTTTC CTGTTATCCC
1501 CTGTCCCAGC CACATCATCC CCATCAGGGT GGGTAATGCA GCACTCAACA
1551 GCAAGATCTG TGATCTTCTG CTCTCCAAGC ACAGCATCTA TGTGCAGGCC
1601 ATCAACTACC CAACTGTGCC TCGTGGTGAG GAGCTACTGC GCTTGGCCCC
1651 CTCCCCCCAC CACAGCCCTC AGATGATGGA AAACTTTGTG GAGAAGCTGC
1701 TGCTGGCCTG GACTGAGGTG GGGCTGCCCC TCCAAGATGT GTCTGTGGCT
1751 GCATGCAACT TCTGTCATCG TCCTGTGCAC TTTGAACTTA TGAGCGAGTG
1801 GGAGCGATCC TACTTTGGGA ACATGGGACC CCAATATGTT ACCACCTATG
1851 CTTAAGGGGA TCCTCTACGC CGGACGCATC GTGGCCGGCA TCACCGGCGC
1901 CACAGGTGCG GTTGCTGGCG CCTATATCGC CGACATCACC GATGGGGAAG
1951 ATCGGGCTCG CCACTTCGGG CTCATGAGCG CTTGTTTCGG CGTGGGTATG
2001 GTGGCAGGCC CCGTGGCCGG GGGACTGTTG GGCGCCATCT CCTTGCATGC
2051 ACCATTCCTT GCGGCGGCGG TGCTCAACGG CCTCAACCTA CTACTGGGCT
2101 GCTTCCTAAT GCAGGAGTCG CATAAGGGAG AGCGTCGATC GACCGATGCC
2151 CTTGAGAGCC TTCAACCCAG TCAGCTCCTT CCGGTGGGCG CGGGGCATGA
2201 CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA ACTCGTAGGA
2251 CAGGTGCCGG CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG
2301 GAGCGCGACG ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG
2351 CCCTCGCTCA AGCCTTCGTC ACTGGTCCCG CCACCAAACG TTTCGGCGAG
2401 AAGCAGGCCA TTATCGCCGG CATGGCGGCC GACGCGCTGG GCTACGTCTT
2451 GCTGGCGTTC GCGACGCGAG GCTGGATGGC CTTCCCCATT ATGATTCTTC
2501 TCGCTTCCGG CGGCATCGGG ATGCCCGCGT TGCAGGCCAT GCTGTCCAGG
2551 CAGGTAGATG ACGACCATCA GGGACAGCTT CAAGGATCGC TCGCGGCTCT
2601 TACCAGCCTA ACTTCGATCA TTGGACCGCT GATCGTCACG GCGATTTATG
2651 CCGCCTCGGC GAGCACATGG AACGGGTTGG CATGGATTGT AGGCGCCGCC
2701 CTATACCTTG TCTGCCTCCC CGCGTTGCGT CGCGGTGCAT GGAGCCGGGC
2751 CACCTCGACC TGAATGGAAG CCGGCGGCAC CTCGCTAACG GATTCACCAC
2801 TCCAAGAATT GGAGCCAATC AATTCTTGCG GAGAACTGTG AATGCGCAAA
2851 CCAACCCTTG GCAGAACATA TCCATCGCGT CCGCCATCTC CAGCAGCCGC
2901 ACGCGGCGCA TCTCGGGCAG CGTTGGGTCC TGGCCACGGG TGCGCATGAT
2951 CGTGCTCCTG TCGTTGAGGA CCCGGCTAGG CTGGCGGGGT TGCCTTACTG
3001 GTTAGCAGAA TGAATCACCG ATACGCGAGC GAACGTGAAG CGACTGCTGC
3051 TGCAAAACGT CTGCGACCTG AGCAACAACA TGAATGGTCT TCGGTTTCCG
3101 TGTTTCGTAA AGTCTGGAAA CGCGGAAGTC AGCGCCCTGC ACCATTATGT
3151 TCCGGATCTG CATCGCAGGA TGCTGCTGGC TACCCTGTGG AACACCTACA
3201 TCTGTATTAA CGAAGCGCTG GCATTGACCC TGAGTGATTT TTCTCTGGTC
3251 CCGCCGCATC CATACCGCCA GTTGTTTACC CTCACAACGT TCCAGTAACC
245
3301 GGGCATGTTC ATCATCAGTA ACCCGTATCG TGAGCATCCT CTCTCGTTTC
3351 ATCGGTATCA TTACCCCCAT GAACAGAAAT CCCCCTTACA CGGAGGCATC
3401 AGTGACCAAA CAGGAAAAAA CCGCCCTTAA CATGGCCCGC TTTATCAGAA
3451 GCCAGACATT AACGCTTCTG GAGAAACTCA ACGAGCTGGA CGCGGATGAA
3501 CAGGCAGACA TCTGTGAATC GCTTCACGAC CACGCTGATG AGCTTTACCG
3551 CAGCTGCCTC GCGCGTTTCG GTGATGACGG TGAAAACCTC TGACACATGC
3601 AGCTCCCGGA GACGGTCACA GCTTGTCTGT AAGCGGATGC CGGGAGCAGA
3651 CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCGCAGC
3701 CATGACCCAG TCACGTAGCG ATAGCGGAGT GTATACTGGC TTAACTATGC
3751 GGCATCAGAG CAGATTGTAC TGAGAGTGCA CCATATGCGG TGTGAAATAC
3801 CGCACAGATG CGTAAGGAGA AAATACCGCA TCAGGCGCTC TTCCGCTTCC
3851 TCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC
3901 AGCTCACTCA AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACG
3951 CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA
4001 AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA
4051 TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT
4101 AAAGATACCA GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT
4151 CCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG
4201 CGTGGCGCTT TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGG
4251 TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC
4301 CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA
4351 CGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA
4401 GGTATGTAGG CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGC
4451 TACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC
4501 CTTCGGAAAA AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTG
4551 GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA
4601 GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG
4651 GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA
4701 TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA
4751 AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA
4801 GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC
4851 TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC
4901 AGTGCTGCAA TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC
4951 AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA
5001 CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA
5051 AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTGCAGG
5101 CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT
5151 CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG
5201 GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT
5251 GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC
5301 CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC
5351 TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAACACG
5401 GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA
5451 AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC
5501 AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC
5551 TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA
5601 AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT
5651 TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA
5701 CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT
5751 TTCCCCGAAA AGTGCCACCT GACGTCTAAG AAACCATTAT TATCATGACA
5801 TTAACCTATA AAAATAGGCG TATCACGAGG CCCTTTCGTC TTCAAGAA
246
APPENDIX D GC AND HPLC METHODS
AZW2.Meth
Figure D-1. AZW2.Meth began with an initial temperature of 140°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.
AZW3.Meth
Figure D-2. AZW3.Meth began with an initial temperature of 100°C for 12 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 5 min.
247
BTS2.Meth
Figure D-3. BTS2.Meth began with an initial temperature of 100°C for 10 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.
BTS3.Meth
Figure D-4. BTS3.Meth began with an initial temperature of 90°C for 20 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.
248
BTS4.Meth
Figure D-5. BTS4.Meth began with an initial temperature of 70°C for 2 min, followed by an increase at 0.3°C/min to a temperature of 90°C which then immediately increased at 20°C/min to temperature of 180°C at which the program remained for 3 min.
BTS7.Meth
Figure D-6. BTS7.Meth began with an initial temperature of 100°C for 30 min, followed by an increase at 20°C/min to a temperature of 180°C at which the program remained for 3 min.
249
BTS8.Meth
Figure D-7. BTS8.Meth began with an initial temperature of 100°C for 15 min, followed by an increase at 20°C/min to a temperature of 195°C at which the program remained for 10 min.
FB1.Meth
Figure D-8. FB1.Meth began with an initial temperature of 60°C for 5 min, followed by an increase at 20°C/min to a temperature of 200°C at which the program remained for 8 min.
250
JON.Meth
Figure D-9. JON.Meth began with an initial temperature of 60°C for 2 min, followed by an increase at 10°C/min to a temperature of 195°C at which the program remained for 10 min.
SEF.Meth
Figure D-10. SEF.Meth began with an initial temperature of 60°C for 5 min, followed by an increase at 10°C/min to a temperature of 195°C at which the program remained for 10 min.
251
YAP.Meth
Figure D-11. YAP.Meth began with an initial temperature of 90°C, followed by an increase at 10°C/min to a temperature of 130°C, followed by an increase at 2°C/min to a temperature of 150°C, followed by an increase at a rate of 20°C/min to a temperature of 250°C at which the program remained for 5 min.
LMM.Meth
Figure D-12. LMM.Meth had a flow rate of 1 mL/min and began with initial conditions of 3% B which were maintained for 2.5 min. This was followed by a linear increase to 18% B over 5 min which was then followed by a linear increase to 28% B over 3.5 min. Next followed a linear increase to 90% B over 10 min. After a 5 min hold at 90% B, a linear decrease to 3% B over 3 min was followed by a 5 min hold at the initial conditions of 3% B.
252
RWP2.Meth
Figure D-13. RWP2.Meth had a flow rate of 1 mL/min and with initial conditions of 0 % B. The method began with an immediate linear increase to 15% B over 15 min, followed by an another increase to 50% B over 15 min, followed by yet another linear increase to 100% B over 4 min at which the program stayed for 3 min. This was followed by a linear decrease to 0% B over 3 min at which the program remained for 10 min.
253
APPENDIX E PLASMID MAPS
pET3b-OYE
Figure E-1. pET3b-OYE.
pBS2
Figure E-2. pBS2.
pET3b-OYE6209 bp
T7 terminator - 404
NdeI - 549 - CA'TA_TG
Trp 116 - 1770
NdeI - 2119 - CA'TA_TG
T7 promoter - 2185
T7 te rminator T7 tag
OY
E
T7 p
r omo t er
bla
pBS27292 bp
T7 term - 72
XhoI - 158 - C'TCGA_G
NdeI - 1402 - CA'TA_TG
XbaI - 2125 - T'CTAG_A
T7 promoter - 2177
OYE2.6-GS
T Fusio
n P
rote
in
bla
T7 term
T 7 pro
mot
e r
254
pFB1
Figure E-3. pFB1.
pRP4
Figure E-4. pRP4.
pFBI6607 bp
T7 term - 26
XhoI - 158 - C'TCGA_G
NdeI - 1402 - CA'TA_TG
T7 promoter - 1474
OYE2.6bla
T7 promo ter
T7 term
pRP46580 bp
T7 term - 26
XhoI - 158 - C'TCGA_G
NdeI - 1375 - CA'TA_TG
T7 promotor - 1447
bla
OYE3
T7 term
T 7 promotor
255
pGF23
Figure E-5. pGF23.
pGF235848 bp
pho A promoter - 229
SalI - 319 - G'TCGA_C
BamHI - 1858 - G'GATC_Cm
AL
AS
bla
pho promoter
256
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BIOGRAPHICAL SKETCH
Robert Wilson Powell III was born in Pensacola Florida in November 28, 1982.
He graduated from Florida A & M University in 2010 with a B.Sc. in Chemistry. He
began studying at the University of Florida in 2011. There he joined Dr. Jon Stewart’s
group and began studying biocatalysis. After which, he spent the next five years running
the reactions that are mentioned in this document.
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