towards new enzymes: from triosephosphate isomerase to kealases
DESCRIPTION
Towards new enzymes: From Triosephosphate Isomerase to Kealases. Peter Neubauer University of Oulu 31.08.2006 (PDB2006). Introduction. Enzyme of the glycolysis pathway W ild type enzyme is a dimer that consists of two identical ( )8-fold subunits with the size of 250 residues - PowerPoint PPT PresentationTRANSCRIPT
Towards new enzymes:
From Triosephosphate Isomerase to Kealases
Peter NeubauerUniversity of Oulu31.08.2006 (PDB2006)
Peter Neubauer
Introduction
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Norledge et al; Proteins: structure, function and genetics (2001)
• Enzyme of the glycolysis pathway
• Wild type enzyme is a dimer that consists of two identical ()8-fold subunits with the size of 250 residues
• Loop-1 and 4 (subunit 1) and loop-3 (subunit 2) form the dimer interfase
• Highly specific binding pocket (phosphate) is formed by loops 6, 7 and 8
• Catalyses the inter-conversion of dihydroxy acetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (DGAP)
Loop6
Loop7
Peter Neubauer
Introduction
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Catalytic residues:Lysine 13Histidine 95Glutamate 167Ligand is represented as green densityLoop-6 closes upon ligand binding
Loop6
Loop7
Catalytic residues:Lysine 13Histidine 95Glutamate 167Ligand is represented as green densityLoop-6 closes upon ligand binding Reaction mechanism as proposed by Kursula et al. Eur.
J. Biochem (2001)
Peter Neubauer
Dimer to monomer
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Borchert et al (1993; 1994)
Thanki et al. (1997)
e.g. Wierenga (2001)
ml8b TIMNorledge etl. (2001)
• Dimeric stabilization is important for the catalytic machinery in wild type TIM
• MonoTIM and ml1 TIM are active enzymes and catalyze the substrates in a similar fashion
• The affinity of the monomeric TIMs for the transition state analogues is lower
• The turnover number for the monomeric TIMs is lower
• Ml8b TIM does no longer convert any known substrates. However the active site remains the same.
Peter Neubauer
Monomeric TIM activity
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Mode of binding of 2PG in wild type and monomeric TIM (ml1 TIM).
Lys 13
2PG
Glu 167
His 95
Loop-6
• The turnover number is 1000 lower in monomeric TIM compared to wild type This is correlated with a different environment, such as an increased solvent accessibility
• The reduction in affinity for 2PG is less than for the phosphate moiety
• Lysine 13 and Histidine 95 show more conformational flexibility compared to wild type. Monomeric TIMs are more “floppy“
• The more accessible active site is an interesting starting point for enzyme engineering
Peter Neubauer
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Tools for new enzymes
What?
• Design of new artificial enzymes
• Design and organic synthesis of new substrates
• Creating active enzymes
ChemistryWild type studies
•iterative process
(Random) mutagenesis
Selection of best mutants
Screen for activity
Pool of enzymes
TIM (variants)[inactive]
KealasesInput Output
Mass Spectrometry
X-Ray crystallography
NMR
Multidisciplinary approach of the biocatalyst consortium
Importance of chirally active aldehydes
Our Start: Our goal:
Wild type studies
Peter Neubauer
Recent wild type studies
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• Investigations on the properties of the conserved active site proline identified a molecular switch to bring the enzyme in a competent state for catalysis upon ligand binding.
• Enzymology studies on P168A mutant versus wild type show a dramatic drop in Kcat/Km
• The affinity for 2PG is reduced • The mode of binding of 2PG in wild type differs from P168A
mutant.• Conclusions:
► Proline 168 is needed for conformational strain around the catalytic glutamate. This strain is transferred to the catalytic glutamate.
► Ligand binding triggers the conformational switch of the catalytic machinery needed for catalysis.
Poster: ”The functional role of the conserved active site proline of triosephosphate isomerase”
Peter Neubauer
Why monomeric TIM?
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Monomeric TIM is a very suitable protein for biocatalysis:
• small size (suitable for NMR; easy to crystallize): so far all constructs of monomeric TIMs could be crystallised)
• easily expressed in high amounts of soluble protein in E.coli
• Monomeric protein has advantages (Vanvaca et al. PNAS
2004) • No cofactors needed• In the closed conformation
the binding site is an extended groove: ml8b TIM (*)
• Its wild type precursor is the extensively studied TIM.
Peter Neubauer
Tailored ligands
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The Concept
• Catalytic head group stays the same
• Achor part contributes most to the binding affinity
• The anchor is optimized for maximum affinity, but can be cleavable
A possible Substrate
A Possible Inhibitor
(cleavable) Anchor Catalytic head group
Original binding site
Catalytic - S i t eExtended binding pocket
Enzyme surface
Peter Neubauer
A-TIM
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• Over 20 constructs based on ml8b TIM have been created
• Several mutations have been investigated: pocket, rim, active site and loop mutants. Many have structures have been solved
• A variant with a deeper and wider binding groove have been created: A-TIM
• A-TIM is as stable as ml1 TIM in solution
• The active site is identical to wild type TIM
Active site
Extended groove
Peter Neubauer
A-TIM
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• Over 35 novel tailored ligands have been created
• Binding studies with NMR, Surface Plasma Resonance and X-ray crystallography have identified lead compounds which can be called “binders”
• Site directed evolution is the next logical step to convert “binders“ to substrates
• Site directed evolution has worked previously to improve activity
• If A-TIM variants converts α-hydroxy ketones to α-hydroxy aldehydes, they can be called Kealases
Saab-Rincon et al. Protein eng. (2001):
Poster: Non-natural enzymes: Directed evolution of a monomeric triosephosphate isomerase variant – a case study
Peter Neubauer
Recent findings
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• In wild type Proline 168 acts as a molecular switch upon ligand binding. Manuscript is submitted
• A study on C-terminal hinge residue A178 in wild type and monomeric TIM provides interesting details about the “floppy“ nature of monomeric TIM. Manuscript is under preparation
• An atomic resolution structure of wild type TIM with PGH (0.82 Å) will give new insights in the reaction mechanism (data under investigation) 1.06 Å detail of A-TIM variant
Peter Neubauer
Recent findings
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Catalytic end
Anchor
OH OC
CC
OP
O
OH O
A R
New binding
Loop 7
Citric acid binding in
A-TIM
Loop 8
O O
C
OO
CC
CC
OO O
CV
A
R
Transition state analogue wild type
Substrate wild type
A R
OHO
OC
CO
P
O
OH
• In X-ray structures the three following compounds are seen as “binders“
• In an X-Ray structure BHAP (bromo hydroxyactone phosphate; a suicide inhibitor) has been found to react in the active site similar to wild type.This finding indicates that A-TIM can still convert the original substrate (Kcat is too low to measure):
• A-TIM is an active molecule
O
O
OH
OH
OH
O
OHS
O
CH3
O
O
O
OHOH
O OH
OH
Citric acid
Malic acid
MW-1
Mare, de La et al. Biochem J (1972)
Peter Neubauer
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• New A-TIM enzymes towards the transformation of new modified nucleosides
• New result: citric acid binds to the active site of A-TIM (structure 1.5 Å)
• Also malic acid and compound MV-1 bind to the active site (seen in X-ray)
• Surface plasma resonance method to screen for binders has been established (Dr. Päivi Pirila)
• Citric acid as positive control binds in the assay specifically
• Other compounds tested (with good results):
WOZ
O
H 2
C
O
O H
H
O H
H
Y X
WOZ
O
H 2
C
OH
O
H
OH
Y X
O
H
O
H
H 2
C
H
O
OHX
Y
WZ
O
1
23
4
5
O
O H
OH
HY
X
O
WZ
O
-/-anomersO
OH
H
OHY
X
O
WZ
O
ribo
arabino
AV-TIMtransformation =
= Anotherpresentation
-/-anomers
Ribo or Arabinodepending on the stereochemistry of the C2=O group reduction
R et roa nalysi s; gene ra l Sc hem eMikhai lopulo I.A .
1 2
3
4 5
Spontaneous cyclization
O
O
OH
OH
OH
O
OHS
O
CH3
O
O
O
OHOH
O OH
OH
A possible application for A-TIM
Citric acid
Malic acid
MW-1
Peter Neubauer
Acknowledgments
Faculty of Science
Department of Biochemistry
Prof. Rik WierengaMarkus Alahuhta
Mikko SalinVille Ratas
Department of Chemistry
Prof. Jouni PursiainenRitva Juvani
Prof. Marja LajunenMatti Vaismaa
Dr. Sampo MattilaNanna Alho
Faculty of Technology
Bioprocess Engineering
Prof. Peter NeubauerDr. Mari YlianttilaMarco Casteleijn
Lilja Kosamo
This work has been supported by the Academy of Finland (project 53923) and
Tekes (project Biocatnuc)
website: http://www.oulu.fi/bioprocess/
Collaborators
University of Kuopio
Department of Pharmaceutical Chemistry
Prof. Seppo Lapinjoki
Technical University of Helsinki
Dr. Petri Pihko
University of Antwerp, Belgium
Prof. Koen AugustynsProf. Anne-Marie Lambeir
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