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Biocatalysis at KTHKarl HultSchool of BiotechnologyRoyal Institute of Technology (KTH)Stockholm, Sweden
Biocatalysis at KTHKarl HultSchool of BiotechnologyRoyal Institute of Technology (KTH)Stockholm, Sweden
To advance the understanding and use of enzyme catalysis
based on a broad interdisciplinary competence in enzymology, chemistry, molecular biology, and
computational chemistry
Vision
Enzyme systems of special interests of the Biocatalysis Group
• Lipases with special interest in Candida antarctica lipase BSubstrate specificityReaction specificityUse in synthesis
• TransaminasesProduction of chiral amines from ketones
Reaction mechanism of lipasesAcylation Deacylation
Acyl-enzyme
Ser105
His224
His224
His224
Oxyanion holeO
O NHN
O
NHHN
NHN
HN
HNHO
Gln106
Thr40
Ser105
HO
O
R*
R''
O
O
R*
R''O
Ser105
O
HO
R*
R''
O
Asp187
O
OAsp187
O
OAsp187
Ser105
His224
His224
His224
Oxyanion holeO
O NHN
O
NHHN
NHN
HN
HNHO
Gln106
Thr40
Ser105
HO
O
R'
R''
O
O
R'
R''O
Ser105
O
HO
R'
R''
O
Asp187
O
OAsp187
O
OAsp187
Lipase B from Candida antarcticais a serine hydrolase belonging to the α/βhydrolase family with a central β-sheetSurrounded by α-helixes
Lipase B from Candida antarctica is very active and stable in most organic solvents
Candida antarctica lipase BCandida antarctica lipase B
The activesite is deep in theproteinstructure
Lipase catalysed kinetic resolution of a chiral alcohol
OHROR
OHR
R'
O
O O
O
R'
OR
O
R'
OEnz
O
R'OHEnz
Thr40
Acyl chain
Large groupof alcohol
Medium groupof alcohol
Stereoselectivity pocket
Leu278
Trp104
Ala281
Ile285
His224
Ser105
R-3-Hexyl octanoatein the active site of CALB wt
The size of the stereoselectivitypocket is set by Trp104
R-3-Hexyl octanoatein the active site of CALB W104A
New volume created by W104A mutation
Ala104
Ser105His224
Enantioselectivity changed 8 300 000 by one point mutation, ΔΔG# 40 kJ/mol.
kcat for the S-enantiomer increased 65 000 times, ΔΔG# 28 kJ/mol
OHThe enantioselectivity changed8 300 000 times by one point mutationafter rational design
Magnusson A, Hamberg A, Takwa M and Hult K, 2005, Angewandte Chemie Int Ed, 44: 4582
Enzyme Enan- KM kcat kcat/KM E tiomer mM s-1 M-1s-1 wt R 61 570 9400 1300000 S 71 0.00053 0.007 W104A R 29 4.4 150 0.15 S 34 34 1000
-40
-20
0
20
40
kJ/m
ol ΔΔGΔΔH -TΔΔS
Enthalpic and entropic contributions to enantioselectivity for CALB W104A
R
S
OH OH OH OH OH
Vallin M, Syrén PO and Hult K, 2010 ChemBioChem 11: 411-416
OH
*
* Butyrate
17 4813 36 100
8
Oxyanion hole Candida antarctica lipase B
Gln106
Thr40
R-2-Butyl octanoatetetrahedral intermediate in the active site of CALB
Oxyanion hole details in Candida antarctica lipase B
O C O-HN NH
O-
O
HO
HN
Ser105OAsp187
His224
+HN
Thr40
Gln106
O C O-HN NH
O-
O
ZHHN
Ser105OAsp187
His224
+HN
Thr40Val
Gln106
Mutation Thr40Ala decreases the activity 2500 times, ΔΔG‡ = 20 kJ/mol
Substrate-assisted catalysis can rescue part of the lost of activityΔΔG‡ = 9 kJ/mol
Magnusson, Hult and Holmquist, 2001, JACS 123, 4354
Substrate assisted catalysis can be used toincrease the yield of monoesters of diols
Patent application with BASF
CALB catalyzes Michael additions
HO
NHO
NH
R'NuH
His
R''
NuH Michael addition
R'SH, R'NHR'', R'CHR''R''' or H2O2
Reaction mechanism for Michael-type addition in CALB Ser105Ala with a thiol
Two-step mechanism suggested by abinitio calculations.
Computed potential energy surface B3LYP/6-31+G* energies (dashed line) relative the reaction complex (1). Solid line represents energies corrected for solvation effects.
O
HHH
H
N
N
H
SH
2TS
O
H
N
N
H
HH
SH
H
N
N
H
H
O
HHH
SH
4TS
O
HHH
S
N
N
H
HH
δ
δ
δ
δ
1 3
N
N
H
OH
H
SH
H
5
2TS 4TS1 3 5
0
10
-10
-20
ΔE /kcal/mol
0.02.0
-13.5
-1.7
-14.1
-15.7-21.2
0.7
-5.6
Reaction Coordinate
Carlqvist, P.; Svedendahl, M.; Branneby, C.; Hult, K.; Brinck, T.; Berglund, P. ChemBioChem, 2005, 6, 331-336.
Diethyl amine reacts very fast with the wild type lipase
lipase
organic solventNH
O
O N
O
O
kappcat 810 min-1
Carlqvist, Svedendahl, Branneby, Hult, Brinck, Berglund, ChemBioChem, 2005, 6, 331-336.
0
20
40
60
80
100
120
0 2 4 6 8 10
Time / min
Prod
uct f
orm
atio
n / %
CALB Ser105AlaCALB wild-typeCarrier without enzymeSubstrates only
Mutant-catalyzed Michael addition of acetyl acetone to methyl vinyl ketone is
extremely fast
kcat,app = 4 000 s-1
Svedendahl, M.; Hult, K.; Berglund, P. JACS 2005, 127, 17988
OCandida antarctica lipase B
Ser105Ala
1:1.4, bulk ,20 °C
O
O
O
O
O
Summary of reactions and rates
Bond Rate (“kcat”) and reaction type min-1
“Natural” C-O transacylation 34 000
“Unnatural” C-C aldolase 0.045 C-O epoxidation 0.9 C-S Michael addition 4.3 C-N Michael addition 810 C-C Michael addition 240 000
Enzyme contributes to catalysis by decreasing transition state energy
Enzyme + substrate
ΔΔG#, enzyme contribution
Transition state, no enzyme
ΔG#enz
Enzyme substratecomplex
Transition statewith enzyme
"KM"
"kcat/KM"
ΔG#no enz
"knon""(kcat/KM)/knon"
Free
ene
rgy,
G
Reaction coordinate
How well is the tentative reactionmechanism using the active site?
Reaction ΔΔG‡ H-bond [kJ mol-1] equivalents
Epoxidation 39 2.0 Michael addition, C-S 42 2.2 Michael addition, C-C 48 2.5
The mutation Thr40Ala showed that one hydrogen bond in the oxyanion hole was worth almost 20 kJ mol-1 . This allows an estimation of the number of hydrogen bonds formed in transition state. The tentative reaction mechanism involves four hydrogen bonds.
Polycondensation with acid end-capping functionalization
B BB B B BA A A A A AB BA A
AA
Catalyst
AAAA
AA
B BB B
B BB B
CACO
OBO OBO
Lipasen RO2CACO2R + (n+1) HOBOH + 2 R'O2CZ
CZO
+ 2n ROH + 2 R'OHO
nZC
O
E‐OHE‐OOC(AB)nOOCZE‐OOC(AB)nOHE‐OOC(AB)nACOOR
E‐OOCZ
R’OOCZR’OH
ZCOOB(AB)n+xOOCZHOB(AB)n+xOOCZHOB(AB)n+xOHROOC(AB)n+xOHROOC(AB)n+xACOORYOOC(AB)m+nACOORROOC(AB)n+xOOCZROOCACOOR
ZCOOB(AB)xOHHOB(AB)xOHROOC(AB)xOHHOBOHROH
ZCOOB(AB)mOHHOB(AB)mOHROOC(AB)mOHHOBOHROH
ZCOOB(AB)m+nOOCZHOB(AB)m+nOHHOB(AB)m+nACOORHOB(AB)m+nOOCZROOC(AB)m+nACOORROOC(AB)m+nOOCZ
ZCOOB(AB)mOOCZHOB(AB)mOOCZROOC(AB)mOOCZ
HOB(AB)mOOCZHOB(AB)mOHROOC(AB)mOH
Cyclic product
Product:ZCOOB(AB)pOOCZ
Substrates:ROOCACOORHOBOHZCOOR’
B BB B B BA A A A A AB BA A
AA
Catalyst
AAAA
AA
B BB B
B BB B
22
H2O excluded
23
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
1500
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z0
500
1000
1500
Inte
ns. [
a.u.
]
500 1000 1500 2000 m/z
20 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z0
500
1000
Inte
ns. [
a.u.
]
1000 1500 2000 m/z
60 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z0
1000
2000
Inte
ns. [
a.u.
]
1000 1500 2000 2500 m/z
120 min
0
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
15000
300
600
Inte
ns. [
a.u.
]
1000 2000 2500 3000 m/z
24 h
1500
MALDI-TOF-MS kinetic study
Dynamic reaction system
NMR of a one step CALB synthesized
telechelic polycondensation polymer
OO
O
OSH
HS
UV induced cross linking affords films
OO
OO
O
O
O
O
O
Om n
10 10
OO
OO
O
O
O
O
O
O
O
On
10
n
OO
O
O
HS SH
7
10
n
OO
O
O
HS
Examples of polycondensation products
Examples of ring opening products
Other polymer products
Materials can be made from one-step synthesiswithout product purificationby UV initiated cross linking
OO
O
O
O
RO
O
O
O
R-OH+ H
OO
O
O
O
RO
O
O
O
HOO
O
O
O
RO
O
O
O
H+
CALB
nn
CALB
Propagation
Initiation
Enzyme catalyzed polylactide synthesis is problematic due to enzymes’ substrate specificity
NNHO
HO
Ser105
OH
O
O
H
H
H
N
O
N
His224
Asp187
O
Gln106
Thr40
O
OO
O
OO
Acyl donor
Acyl acceptor
Intermediate used for molecular modelling
Important residues for mutation were identified by molecular modelling
Enzyme Initiation Propagation Rate (s-1) WT 40 1 Q157A 180 93 Q157A, I189A, L278A 770 83
VINNOVA projectPer Berglund
Rational design of ω-transaminase from Arthrobacter citreus
• Aim: Increase the enantioselectivity for phenylacetone-type of substrates (98% ee)
• Homology model needed• Docking of pro-S and pro-R quinonoid
NH2 OF O F
NH2
Rational design of ω-transaminase from Arthrobacter citreus
• Homology model needed
Rational design of ω-transaminase from Arthrobacter citreus
Docking of pro-S and pro-R quinonoid
Rational design of ω-transaminase from Arthrobacter citreus
CNB05-01 or variantsNH2 O
F O FNH2
Mutant ee Enantiomer Enantiomeric % ratio Wt 98 S 100 Tyr331Cys 99,5 S 400 Val328Ala 58 R 4
Many good scientist students and friends have contributed to this work but most of all members of the Biocat and BioPol Groups at KTH
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