directed evolution.pdf - Department of Chemistry – Colorado State ...

Bocola, M. et al. Adv. Synth. Catal. 2005, 347, 979.
“Directed Evolution” in Enantioselective
Enzymatic Catalysis
Todd K. Hyster
Third Year Seminar
November 22, 2010

Enzyme Catalysis
Enzyme Starting Material Product/
Starting Material/
Enzyme Product
Enzyme Complex
Enzyme Complex
- Benefits
- “Green” Alternative
- High Selectivity
H 2N
O
R 1 Ph
OEt
Pig Liver Esterase
H 3N
- Drawbacks
- Solvent Variability
- Thermal Stability
- High Selectivity
R = Allyl, Bu = >95% ee, 31% conv.
All Others

How to Solve Selectivity Problems?
Transition Metal
Catalysis
Ligand
Development
Bio-Catalysis
Organocatalysis
Catalyst
Development

Basic Biochemistry - Gene and Enzyme
Gene - DNA which codes for a specific polypeptide
O O
P
O
O
H
O
O
N
NH 2
N
Cytosine
O
HN
O
N
Thymine
H 2N
N
O
N
Guanine
Enzyme - A complex polypeptide with a unique structure
Alanine
Aspartic Acid
Cysteine
Glutamic Acid
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
Leucine
O
Base
Methionine
Asparagine
Proline
Glutamine
Arginine
Serine
Threonine
Valine
Tryptophan
Tyrosine
Me
Ala
Gln
Asn
Ile
Val
Glu
Arg
Gly
His
N
N
N
NH 2
N
Adenine
Bocola, M. et al. Adv. Synth. Catal. 2005, 347, 979. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th
Ed.; W H Freeman, New York, 2002.
N
N

Basic Biochemistry - Transcription and Translation
• Transcription and Translation - The process of transforming DNA into
Enzymes
DNA RNA Protein
Use RNA
polymerase
Bocola, M. et al. Adv. Synth. Catal. 2005, 347, 979. Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th
Ed.; W H Freeman, New York, 2002.
Codon

Reetz, M. T. J. Org. Chem. 2009, 74, 5767.
Repeat
Directed Evolution
gene (DNA) wild-type enzyme
random
mutagenesis
library of mutated genes
expression
library of mutated enzymes
screening for
the desired trait
positive mutant

Directed Evolution v. Rational Design
Repeat
gene (DNA) wild-type enzyme
random
mutagenesis
library of mutated genes
expression
library of mutated enzymes
screening for
the desired trait
positive mutant
Important Site for
Structure
Ala
Gln
Asn
Ile
Val
Glu
Arg
Gly
His
Ala
Gln
Asn
Met
Val
Glu
Arg
Gly
His
- structural knowledge not necessary
Reetz, M. T. J. Org. Chem. 2009, 74, 5767. Arnold, F. H.; Acc. Chem. Res. 1998, 31, 125. Cedrone, F. et al. Curr. Opin. Struct. Biol. 2000, 10, 405.

Timeline of Directed Evolution
1960 2000
Spiegelman preformed an
extracelluar evolutionary
experiment with a selfduplicating
nucleic acid”
(Mills, D. R.; Peterson, R. L.;
Spiegelman, S. PNAS 1967,
58, 217.)
Hansche coined the term
“Directed Evolution”
(Francis, J. C.; Hansche, P.
E. Genetics 1972, 70,
59.)
Eigen suggests a
mechanism for
molecular
evolution.
(Eigen, M.;
Gardiner W. PNAS
1984, 56, 967.)
A team from
Synergen used
iterative rational
mutagenesis
(Liao, H.;
McKenzie, T.;
Hageman, R. PNAS
1986, 83, 576.)
Arnold reports the first
iterative mutagenesis to
tune enzyme activity.
(Chen, K.; Arnold, F. H.
PNAS 1993, 90, 5618.)
Reetz reports the first
iterative mutagenesis to
tune enzyme
enantioselectivity.
(Reetz, M. T. et al. ACIE
1997, 36, 2830.)

Mutagenesis
- Error Prone Polymerase Chain Reaction (epPCR)
- Saturation Mutagenesis
- Cassette Mutagenesis
- DNA Shuffling
Reetz, M. T. Directed Evolution of Enantioselective Enzymes as Catalysts for Organic Synthesis In Advances in Catalysis 2006, pp. 1-69.

Mutating Enzymes
- Error Prone Polymerase Chain Reaction (epPCR)
- Polymerase Chain Reaction
- epPCR
- Modify the concentration of MgCl2, MnCl2, or
nucleotides to induce mutations.
N
- Mutations throughout the enzyme
- Some amino acids favored over others
N N H N
N
HN H O
Guanine
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002. Cadwell,
R. C.; Joyce, G. F. Genome Res. 1992, 2, 28.
O
H
NH
N
Me
Cytosine
N
HN
N N H
N
N
O
Adenine
H
O
N
Me
Thymine

Codon Degeneracy
- Error Prone Polymerase Chain Reaction (epPCR)
Second Nucleotide T C A G
First Nucleotide
T TTT Phenylalanine TCT Serine TAT Tyrosine TGT Cysteine
TTC Phenylalanine TCC Serine TAC Tyrosine TGC Cysteine
TTA Leucine TCA Serine TAA Stop TGA Stop
TTG Leucine TCG Serine TAG Stop TGG Tryptophan
C CTT Leucine CCT Proline CAT Histidine CGT Arginine
CTC Leucine CCC Proline CAC Histidine CGC Arginine
CTA Leucine CCA Proline CAA Glutamine CGA Arginine
CTG Leucine CCG Proline CAG Glutamine CGG Arginine
A ATT Isoleucine ACT Threonine AAT Asparagine AGT Serine
ATC Isoleucine ACC Threonine AAC Asparagine AGC Serine
ATA Isoleucine ACA Threonine AAA Lysine AGA Arginine
ATG Methionine ACG Threonine AAG Lysine AGG Arginine
G GTT Valine GCT Alanine GAT Aspartic Acid GGT Gylcine
GTC Valine GCC Alanine GAC Aspartic Acid GGC Gylcine
GTA Valine GCA Alanine GAA Glutamic Acid GGA Gylcine
GTG Valine GCG Alanine GAG Glutamic Acid GGG Gylcine
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.

Mutagenesis
- Saturation Mutagenesis and Cassette Mutagenesis
- Saturation Mutagenesis
replaces one amino
acid with the other 19 amino
acids to form 19 new
mutants
CTGGCCCACGGCATTATTGGCTT
CTGGCCCACGGCGCTATTGGCTT
- Cassette Mutagenesis
replaced a short segment
new amino acids, essentially
saturation mutagenesis over
multiple positions
CTGGCCCACGGCATTATTGGCTT
CTGGCCCATGGCGCTATTGGCTT
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.
Kegler-Ebo, D. M.; Docktor, C. M.; DiMaio, D. Nucleic Acids Res. 1994, 11, 1593.

TTC
AAG
TTC
AAG
Plasmid
Methylated
PCR Site Mutagenesis
TTA
AAT
i) Denature
ii) Anneal
Mutated Primers
Dpn1
for
purification
AAT
TTC
TTC
AAG
Mutated Primers
TTA
AAG
Multiple Rounds
of PCR
TTA
AAT
Zheng, L.; Baumann, U.; Reymond, J.-L. Nucl. Acids Res. 2004, 32, 115. Hutschison, C. A.; Philipps, S.; Edgell, M. H.; Gillham, S.; Jahnke, P.;
Smith, M. J. Biol. Chem. 1978, 253, 6551.

Cassette Formation
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.

- DNA Shuffling
Mutagenesis
mutation
fragmentation
recombination
wild-type
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.
Cohen, J. Science, 2001, 237, 5528.

Reetz, M. T. Angew. Chem. Int. Ed. 2010, Early View.
Directed Evolution Challenges
Maximum Degree of Diversity
N=19 M X!/(X-M)!M!
where:
N = number of mutants
X = number of amino acids in the enzyme
M = number of amino acids substitutions
so in a 300 amino acid enzyme...
If M=1, N=5700 mutants
If M=2, N=16,190,850
If M=3, N=30,557,530,900

C 8H 17
Me
O
O
NO 2
Lipase / E Values
H 2O
Pseudimonas aeruginosa Lipase
285 Amino Acids so...
WT Enzyme Selectivity Factor
one mutation = 5415 mutants
E = 1.1
Starting Material ee% Product ee%
E Values
ln [1-c(1+ee(P))]
ln [1-c(1-ee(P))]
c = conversion
P = product
= E
rate of fast enant.
= E
rate of slow enant.
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830. C.-S Chen, Y Fujimoto, G
Girdaukas and C.J Sih, J. Am. Chem. Soc. 1982, 104, 7294–7299
C 8H 17
Me
O
OH
C 8H 17
For product and starting material with 90% ee @ 50% conversion E = 59
Me
O
O
NO 2

Absorbance
Absorbance
C 8H 17
Me
O
O
t(s)
Lipase / p-Nitrophenol Screen
NO 2
Low E Value
C 8H 17
High E Value
C 8H 17
H 2O
Pseudimonas aeruginosa Lipase
Me
(S)-enantiomer
C 8H 17
(R)-enantiomer
O
O
OH
OH
Me
(S)-enantiomer
(R)-enantiomer
C 8H 17
t(s)
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830.
Me
C 8H 17
O
Me
OH
O
OH
Mutant A
Mutant B
Mutant C
Mutant D
Mutant E
Mutant F
Mutant G
Me
O
OH
C 8H 17
Me
O
O
NO 2
O NO 2 UV = 405 nm
S
S
R
S R
S
S
S
S
R
R
R
R
R
S
S
R
S R
S
S
S
S
R
R
R
R
R
Mutant H
Mutant I
Mutant J
Mutant K
Mutant L
Mutant M
Mutant N

C 8H 17
desired gene
Me
O
epPCR
O
NO 2
desired mutant DNA
Lipase / epPCR
H 2O
Pseudimonas aeruginosa Lipase
express in
lipase deficient
E. coli
1000 - 2400 mutants/generation
desired mutant enzyme
C 8H 17
Screen for
enantioselectivity
repeat x 4
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830. Reetz, M. T. “Directed Evolution of
Enantioselective Enzymes as Catalysts for Organic Synthesis” Advances in Catalysis; Elsevier, 2006.
Me
O
OH
C 8H 17
Me
Run on Chiral
GC/MS
O
O
best mutant enzyme
NO 2
best mutant DNA

C 8H 17
E
Me
O
O
1.1
Lipase Gen. 1 Directed Evolution
2.1
NO 2
4.4
H 2O
Pseudimonas aeruginosa Lipase
9.4
11.3
0 1 2 3 4 5
mutant generation
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830.
Liebeton, K.; Zonta, A.; Schimossek, K.; Nardini, M.; Lang, D.; Dijkstra, B. K.; Reetz, M. T.; Jaeger, K.-E. ChemBiol 2000, 7, 709.
WT
S149G
C 8H 17
Me
O
S155L + S149G
OH
C 8H 17
Me
O
O
NO 2
F259L + V47G + S155L + S149G
V47G + S155L + S149G
20 - 30 % conversion

Pseudimonas Aeruginosa Lipase
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830.
Liebeton, K.; Zonta, A.; Schimossek, K.; Nardini, M.; Lang, D.; Dijkstra, B. W.; Reetz, M. T.; Jaeger, K.-E. Chem. Biol. 2000, 7, 3591.

Koshland Induced Fit Theory
-Emil Fischer initially proposed that substrates and enzymes fit together in a lock and key fashion.
-Daniel Koshland later modified this theory with the argument that enzymes were not rigid
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.
Koshland, D. E. Angew. Chem. Int. Ed. 1994, 33, 2375.

C 8H 17
E
Me
Lipase Gen. II - Saturation Mutagenesis
O
O
WT 1.1
149G 2.1
NO 2
155L 4.4
H 2O
Pseudimonas aeruginosa Lipase
47G 9.4
11.3 259L
0 1 2 3 4 5
mutant generation
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830.
Liebeton, K.; Zonta, A.; Schimossek, K.; Nardini, M.; Lang, D.; Dijkstra, B. K.; Reetz, M. T.; Jaeger, K.-E. ChemBiol 2000, 7, 709.
WT
S149G
C 8H 17
Me
O
S155L + S149G
OH
C 8H 17
Me
O
O
NO 2
S155L + S149G + V47G + F259L
S155L + S149G + V47G

C 8H 17
Me
Lipase Gen. II - Saturation Mutagenesis
O
WT
O
149G
NO 2
155L
H 2O
Pseudimonas aeruginosa Lipase
47G
259L
Reetz, M. T.: Zonta, A.; Schimossek, K.; Liebeton, K.; Jaeger, K.-E. Angew. Chem. Int. Ed. 1997, 36, 2830.
Liebeton, K.; Zonta, A.; Schimossek, K.; Nardini, M.; Lang, D.; Dijkstra, B. K.; Reetz, M. T.; Jaeger, K.-E. ChemBiol 2000, 7, 709.
C 8H 17
Me
O
OH
C 8H 17
Me
O
O
NO 2

C 8H 17
Me
Lipase Gen. II - Saturation Mutagenesis
O
O
- Each mutation represents a
“hot spot”
- Saturation mutagenesis
preformed at each “hot spot”
E
NO 2
30
25
20
15
10
5
1
H 2O
Pseudimonas aeruginosa Lipase
259L
47G
155L
149G
WT
epPCR sat. 155F epPCR
Liebeton, K.; Zonta, A.; Schimossek, K.; Nardini, M.; Lang, D.; Dijkstra, B. K.; Reetz, M. T.; Jaeger, K.-E. ChemBiol 2000, 7, 709.
Berg, J. M.; Tymoczko, J. L.; Stryer, L. Enzymes: Basic Concepts and Kinetics. Biochemistry, 5th Ed.; W H Freeman, New York, 2002.
C 8H 17
Me
O
OH
164G
55G
C 8H 17
Me
O
O
NO 2

C 8H 17
Me
O
Reversing Selectivity - DNA Shuffling
O
NO 2
Can the natural selectivity be reversed?
wild-type enzyme
E=0.9
Gene A
E=1
Zha, D.; Wilensek, S.; Hermes, M.; Jaeger, K.-E.; Reetz, M. T. Chem. Commun. 2001, 2664.
H 2O
Pseudimonas aeruginosa Lipase
Gene B
E=1.1
epPCR 2-3 mutations
Gene C
E=2.0
Gene D
E=3.0
epPCR 2-3 mutations
Gene E
E=3.7
C 8H 17
Me
O
OH
C 8H 17
epPCR 2-3 mutations
Me
O
Gene F
E=7.0
O
NO 2
no further
improvement

C 8H 17
Me
O
Reversing Selectivity - DNA Shuffling
O
NO 2
Can the natural selectivity be reversed?
wild-type enzyme
E=0.9
Gene A
E=1
Zha, D.; Wilensek, S.; Hermes, M.; Jaeger, K.-E.; Reetz, M. T. Chem. Commun. 2001, 2664.
H 2O
Pseudimonas aeruginosa Lipase
Gene B
E=1.1
epPCR 2-3 mutations
Gene C
E=2.0
Gene D
E=3.0
epPCR 2-3 mutations
Gene E
E=3.7
C 8H 17
Me
O
OH
C 8H 17
epPCR 2-3 mutations
Me
O
Gene F
E=7.0
O
NO 2
no further
improvement

C 8H 17
Me
O
Reversing Selectivity - DNA Shuffling
O
NO 2
Can the natural selectivity be reversed?
Gene A
E=1
Gene G
E=6.5
Gene B
E=1.1
Gene K
E=30
Zha, D.; Wilensek, S.; Hermes, M.; Jaeger, K.-E.; Reetz, M. T. Chem. Commun. 2001, 2664.
H 2O
Pseudimonas aeruginosa Lipase
DNA Shuffling
Gene C
E=2.0
Gene H
E=7
epPCR
high mutation rate
Gene E
E=3.7
Gene J
E=20
C 8H 17
Me
Gene I
E=6.7
O
OH
Gene F
E=7.0
C 8H 17
Me
O
O
Gene D
E=3.0
Two mutations in “Gene I” are eliminated
NO 2

C 8H 17
Me
Cohen, J. Science 2001, 237, 5528.
O
Reversing Selectivity - DNA Shuffling
O
NO 2
H 2O
Pseudimonas aeruginosa Lipase
DNAse I
digest
C 8H 17
Me
DNA ligase
PCR w/o
primers
O
OH
C 8H 17
Me
O
O
NO 2

C 8H 17
Best Prior
Mutant
E=25
Me
O
Lipase Gen. III - Cassette Mutagenesis
O
NO 2
1 cycle of high
mutation epPCR
enzyme variants
A with E=3
B with E=6.5
DNA Shuffling
enzyme variant C
E=32
H 2O
Pseudimonas aeruginosa Lipase
wild-type
E=1.1
enzymes variant C
E=30
Reetz, M. T.; Wilensek, S.; Zha, D.; Jaeger, K.-E. Angew. Chem. Int. Ed. 2001, 40, 3589.
C 8H 17
CMCM
Pos. 160 - 163
DNA Shuffling
enzymes variant H
E>51
Me
O
OH
C 8H 17
Me
O
O
CMCM
Pos. 155 + 162
enzymes variants
D with E=34
E with E=30
NO 2

Mutations in s-selective mutatant E=51:
20, 53, 155, 162, 180, 234
Location of Mutations
Catalytic Triad:
82, 229, 251
Mutations in r-selective mutatant E=30:
16, 34, 86, 87, 94, 113, 147, 150, 208, 232, 237
Bocola, M.; Otte, N.; Jaeger, K.-E.; Reetz, M. T.; Theil, W. ChemBioChem 2004, 5, 214. Reetz, M. T.et al. ChemBioChem 2007, 8, 106.

C 8H 17
Me
O
O
NO 2
Catalytic Triad
H 2O
Pseudimonas aeruginosa Lipase
PAL Enzyme Catalytic Triad PAL Enzyme Catalytic Triad with Inhibitor
Bocola, M.; Otte, N.; Jaeger, K.-E.; Reetz, M. T.; Theil, W. ChemBioChem 2004, 5, 214. Reetz, M. T.et al. ChemBioChem 2007, 8, 106.
C 8H 17
Me
O
OH
C 8H 17
Me
O
O
NO 2

O
N
H
R
O
Me
N
H
O
O
Ser 82
R
Me
OR'
O
N
H
H N N
O
O
Ser 82
OH
His 251
O
N
H
H N N
His 251
H O
H O
Mechanism
Bocola, M.; Otte, N.; Jaeger, K.-E.; Reetz, M. T.; Theil, W. ChemBioChem 2004, 5, 214.
Reetz, M. T.et al. ChemBioChem 2007, 8, 106.
O
Asp 229
O
Stereoselective Step
Asp 229
O
N
H
O
R
Me
N
H
R
Me
O
O
Ser 82
O
O
OR'
- HOR
+ H 2O
O
Ser 82
H
O
N
H
N N
His 251
N
H
O H
H
N N
His 251
H
H
O
O
O
Asp 229
O
Asp 229

Origin of Selectivity
Bocola, M.; Otte, N.; Jaeger, K.-E.; Reetz, M. T.; Theil, W. ChemBioChem 2004, 5, 214.
R-enantiomer interacts with 162Leu favoring S-enantiomer

Combinatorial Active-Site Saturation Test (CAST)
-Libraries contain 1-3 amino acids
- Marriage of saturation mutagenesis, random
mutagenesis, and rational design
-High probability of success because the mutants
are near the binding pocket.
Reetz, M. T. J. Org. Chem. 2009, 74, 5767. Reetz, M. T.; Bocola, M.; Carballeira, J. D.; Zha, D.; Vogel, A. Angew. Chem. Int. Ed. 2005, 44, 4192.

Lib. B
Combinatorial Active-Site Saturation Test (CAST)
Lib. D
Lib. C
Lib. E
Lib. A
Mutants at lib. A and lib. D have greatest
effect
C 8H 17
Reetz, M. T.; Bocola, M.; Carballeira, J. D.; Zha, D.; Vogel, A. Angew. Chem. Int. Ed. 2005, 44, 4192.
Reetz, M. T.; Carballeira, J. D.; Peyralans, J.; Höbenreich, H.; Maichele, A.; Vogel, A. Chem. Eur. J. 2006, 12, 6031.
Me
O
O
Me
OR
OR
O
OR
i-Bu
n-Bu
Et
O
O
Me
OR
OR
O
OR
Et
O
O
O
OR
OR
OR
Leu162Val E = 20
Met16Ala, Leu17Phe E = 25

Reetz, M. T. J. Org. Chem. 2009, 74, 5767.
Iterative Saturation Mutagenesis (ISM)
B C D A C D A B D A B C
A B C D
WT
- The marriage of CASTing and directed evolution
- One of the fastest ways to develop a catalyst

RO
O
Me
RO
Iterative Saturation Mutagenesis
O
Et
RO
Me
E = 31 E = 106 E = 55
O
Reetz, M. T.; Prasad, S.; Carballeira, J. D.; Gumulya, Y.; Bocola, M. J. Am. Chem. Soc. 2010, 132, 9144.
i-Bu
RO
O
Me
E = 594
C 8H 17
Met16Ala/Leu17Phe/Leu162Asn E = 594
Met16Ala/Leu17Phe E = 2.6
Met16Ala/Leu162Asn E = 2.2
Leu17Phe/Leu162Asn E = 1.1

The Best of Both Worlds
Mutations at 162 and 16 open the active pocket increasing activity
Reetz, M. T.; Prasad, S.; Carballeira, J. D.; Gumulya, Y.; Bocola, M. J. Am. Chem. Soc. 2010, 132, 9144.

Asp 192
O
His 374
O
N
NH
O
O
H
O
O
Tyr 314
H
O
NO 2
Asp 348
Isotopic Screening
O
(S)
PhO H2O C 6D 5O
O
Tyr 251
Asp 192
His 374
O
OH
H
N
NH
Reetz, M. T. et al. Org. Lett. 2004, 6, 177. Zou, J. Y. et al. Structure 2000, 8, 111.
(R)
Aspergillus niger PhO
O
O
O
O H H
O
Tyr 314
O
NO 2
Asp 348
HO
OH
WT E = 4.6
Mutant E = 10.8
Tyr 251
Asp 192
His 374
O
(S) C 6D 5O (R)
OH
O
O
H
HN
N
O
O
O H H
OH
Tyr 314
O
NO 2
Asp 348
Tyr 251

Epoxide Hydrolase
Reetz, M. T. et al. Org. Lett. 2004, 6, 177. Zou, J. Y. et al. Structure 2000, 8, 111.

Epoxide Hydrolase
Reetz, M. T. et al. Org. Lett. 2004, 6, 177. Zou, J. Y. et al. Structure 2000, 8, 111.

Epoxide Hydrolase - ISM
Reetz, M. T. et al. Angew. Chem. Int. Ed. 2006, 45, 1236. Zou, J. Y. et al. Structure 2000, 8, 111.
B C D A C D A B D A B C
A B C D
WT

Library A
193/195/196
PhO
Library B
215/217/219
Reetz, M. T. et al. J. Am. Chem. Soc. 2009, 131, 7334.
Reetz, M. T. et al. Angew. Chem. Int. Ed. 2006, 45, 1236.
Epoxide Hydrolase - ISM
O
H 2O
Epoxide Hydrolase
Library C
329/330
WT E = 4.6
Library D
349/350
Library E
317/318
Library F
244/245/249
no enhancement E = 14 no enhancement not tested not tested not tested
PhO
O
PhO
HO
OH

no enhancement
Library A
193/195/196
Library F
244/245/249
PhO
Reetz, M. T. et al. J. Am. Chem. Soc. 2009, 131, 7334.
Reetz, M. T. et al. Angew. Chem. Int. Ed. 2006, 45, 1236.
Epoxide Hydrolase - ISM
O
WT E = 4.6
Library E
317/318
H 2O
Epoxide Hydrolase
Library B
215/217/219
E = 49
PhO
Library C
329/330
Library D
349/350
E = 35
O
E = 14
E = 21
E = 24
PhO
HO
Library F
244/245/249
OH
Library E
317/318
E = 115

Asp 192
O
O
d
O
H
O
Tyr 314
H
O
Tyr 251
Epoxide Hydrolase
mutant d R d S !d R-S E
WT 4.3 3.5 0.8 4.6
mA 4.8 4.0 0.8 14
mB
4.9 4.0 0.9 21
mC 5.1 4.0 1.1 24
mD
Reetz, M. T. et al. J. Am. Chem. Soc. 2009, 131, 7334.
5.1 3.9 1.2 35
mE 5.4 3.8 1.6 115

only substrate tolerated
Phenylacetone Monooxygenase
Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
Reetz, M. T.; Wu, S. Chem. Commun. 2008, 5499.
O
Ph
O 2
PAMO, NADPH
O
O Ph

H 2O
Me
Understanding Selectivity - Mechanism
R
N
Me N
Me
NADPH
N O
O
NH
NADP
R
N
Me N
H
O
Reetz, M. T. et al. Angew. Chem. Int. Ed. 2006, 71, 8431.
O
O
N O
O
NH
Me
R
N
Me N H
N O
O
NADP
NH
Me
O 2
Me
Me N
H O
O O
R
N
Me N
H O
NADP O
NADP
R
N
N O
O
NH
Criegee Intermediate
N O
O
NH
O

only substrate tolerated
Phenylacetone Monooxygenase
Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
Reetz, M. T.; Wu, S. Chem. Commun. 2008, 5499.
O
Ph
O 2
PAMO, NADPH
O
O Ph

Parent Sequence at the Bulge:
Ser/Ala/Leu/Ser
Mutant Sequence at the Bulge:
Ala/Trp/Tyr/Thr
when Ar = Ph E = 70
when Ar = C6H4Cl E > 200
Phenylacetone Monooxygenase
Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
Reetz, M. T.; Wu, S. Chem. Commun. 2008, 5499.
O
Ar
O 2
PAMO, NADPH
O
O
Ar
O
Ar

Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
Only the Bulge
Only residues on the “bulge” increased ee and substrate tolerance
proline is thought it impart rigidity

Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
O
Et Mutant, NADPH
Position 440
O 2
mutant E-value Km (mM) kcat (s -1 ) kcat/Km
Phe 26 0.89 1.2 1300
Leu >200 1.6 0.72 450
Ile >200 2.7 0.66 240
Asn >200 2.2 1.5 680
His 34 1.0 0.83 830
Trp >200 1.3 1.3 1000
Tyr 95 1.9 1.1 580
O
O
Et
O
Et

O
O
O
O
O
O
Ph
C 6H 4Cl
C 6H 4Me
Me
Et
n-Pr
Reetz, M. T.; Wu, S. J. Am. Chem. Soc. 2009, 131, 15424.
Scope and Selectivity
E-value E-value
12
7
117
95
26
145
O
O
O
O
O
O
n-Bu
Me
Cy
Bn
Me
CN
39
102
>200
145
48
91

Mutations distant from the active site
No Activity with WT
400 mutants screened
Allosteric Effects
Wu, S.; Acevedo, J. P.; Reetz, M. T. P. Natl. Acad. Sci. USA 2010, 107, 2775.
Gln93Asn/Pro94Asp best mutant
O
O
O
O
Ph
n-Pr
Me
Bn
Me
conv. (%) E-value
45 92
37 68
15 >200
43 >200

Mutations distant from the active site
No Activity with WT
400 mutants screened
Allosteric Effects
Wu, S.; Acevedo, J. P.; Reetz, M. T. P. Natl. Acad. Sci. USA 2010, 107, 2775.
Gln93Asn/Pro94Asp best mutant
O
Me
O
Et
O
n-Bu
ee%
98
98
97

X
X
O
X
X
R
O
H
O
Y
H
Y
Y
O
O
Y
Nitrilase
Baeyer-Villigerase
Aminotransferase
Aldolase
Enoate Reductase
X
X
O
O
O
NH 2
Y
X Y
R
X
OH
O
Y
Conclusion
Y
OH
R
X
X
X
HN
H
O
O
O
Y
O
Y
OR
NH
O
P
RO OR
OR
P450 Oxidase
Lipase
X
Hydantoinase
Epoxide Hydrolase
Kinase
O
OH
R
HN NH 2
X
X
O
O
OH
OH
O
OH
R
Y
Y
OH
OH
P
RO OH
OR

C 8H 17
PhO
O
Me
O
O
O
NO 2
H 2O
Epoxide Hydrolase
Et Mutant, NADPH
O 2
Conclusion
H 2O
Pseudimonas aeruginosa Lipase
PhO
O
PhO
HO
WT E = 4.3
Mutant E = 115
O
O
Et
WT E = 12.8
Mutant E = 200
OH
C 8H 17
References include more of directed evolution in enzyme catalysis
O
Et
Me
O
OH
C 8H 17
WT E = 1.1
Mutant E = 594
Me
O
O
NO 2
- Directed evolution can increase
the enantioselectivity and
substrate selectivity of enzyme
catalysis
- CASTing and ISM represent
new method to rapidly develop
enzymatic catalysis

Acknowledgements
Rovis Group
Tomislav Rovis

E + S
k1
k-1
Enzyme Kinetics
kcat
E:S E:P
Steady State Kinetics
kcat = turnover number
Km = Michaelis Constant (kcat + k-1)/k1
if k-1 >> kcat then Km = Kd = k-1/k1
if kcat >> k-1 then Km = kcat/k1
kcat/Km = Specificity Constant
Anslyn, E. V.; Dougherty, D. A. Modern Physical Organic Chemistry, 1st ed.; University Science Books: Sausalito, 2006.
P

N
NH 2
N
N
O
P O
OH
O
N
O
OH
NADP
O O
O O P
O P O
O
H
HO
O
N
OH
O
NH 2
NADPH
N
NH 2
N
N
O
P O
OH
O
N
O
OH
NADPH
O O
O O P
O P O
O
H
HO
O
N
OH
H H
O
NH 2

NC
OH
CN
WT = 94.5 %ee @ 100 mM
87.8 %ee @ 2.25 M
Nitrilase
A190H
Is this really directed evolution...?
HO 2C
OH
m/z = 130
CN 15
NC
Nitrilases
OH
Using Gene Site
Saturation
Mutagenesis
(GSSM):
S-selective
Nitrilase
NC
DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476. DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024.
CO 2H
PhHN
Ala190His 97.9 %ee @ 100 mM
OH
CN 15
98.1 %ee @ 2.25 M
R-selective
Nitrilase
Ph
O
NC
N
i-Pr
F
Lipitor
OH
m/z = 129
CO 2H
OH
OH CO2H

O
Ph
Eliminate Residues
441+442
Rational Design - Remove the Bulge
O
Ph
O O
Bocola, M.; Schulz, F.; Leca, F.; Vogel, A.; Fraaije, M. W.; Reetz, M. T. Adv. Synth. Catal. 2005, 347, 979.
Ph
O
Ph
O
O

Epimerizable
D-Hydantionase
ee = 40%
MeS
R H
HN NH
Hydantoinase
May, O.; Nguyen, P. T.; Arnold, F. H. Nature Biotechnology 2000, 18, 317.
O
O
2 round of epPCR
HN NH
O
O
Hydantoinase
Mutant Hydantoinase 100 mM
Wild Hydantoinase 100 mM
R H
O
OH
+ H 2O HN NH 2
O
Lower D-Selectivity
Higher Activity
hydantoinase
racemace
L-carbamoylase
MeS
Carbamoylase
R H
O
+ H 2O NH 2
91 mM
66 mM
OH
saturation mutagenesis
O
NH 2
AA95
OH
L-Hydantionase
ee = -20%

O
OH
O 2
CHMO Mutants
Baeyer-Villiger Monooxygenase
O
O
HO
OMe
O
(R)
O
OH
O
(S)
O 2
CHMO Mutant
F432S
O O
(R)
9% ee (R)
O O
(S)
Reetz, M. T.; Brunner, B.; Schneider, T.; Schulz, F.; Clouthier. C. M.; Kayser, M. M. Angew. Chem. Int. Ed. 2004, 43, 4075/
O
H
O
MeO (S)
H
99% ee (S)
OH
OH
Round 1
epPCR
F342L
49% ee
F342S
79% ee
Round 2
epPCR
90% ee

O
O
OH
OMe
O 2
CHMO Mutants
O 2
CHMO Mutant
F432S
Baeyer-Villiger Monooxygenase
O
O
MeO (S)
99% ee (S)
O
HO
O
(R)
O
OH
O
(S)
O O
(R)
9% ee (R)
O O
(S)
Reetz, M. T.; Brunner, B.; Schneider, T.; Schulz, F.; Clouthier. C. M.; Kayser, M. M. Angew. Chem. Int. Ed. 2004, 43, 4075.
Mihovilovic, M. D.; Rudroff, F.; Winninger, A.; Schneider, T.; Schulz, F.; Reetz, M. T. Org. Lett. 2006, 8, 1221.
O
H
H
WT 89% ee (S)
S 94% ee (S)
R 17% ee (R)
O
Me Me
WT 92% ee (S)
S 99% ee (S)
R 99% ee (R)
OH
OH
Round 1
epPCR
54% ee
79% ee
Ph
Round 2
epPCR
90% ee
O
WT 62% ee (S)
S 96% ee (S)
R 8% ee (R)
O
WT No Conv.
S 92% ee (S)
R 12% ee (R)

O
O
O
O
O
O
H
H
H
H
OH
OH
OH
OMe
Me
i-Pr
Et
Understanding Selectivity - Mechanism
WT (ee%) F432S (ee%)
9 R
78 S
95 S
92 S
94 R
27 R
79 S
99 S
99 S
99 S
97 R
97 R
Kayser, M. M.; Clouthier, C. M. J. Org. Chem. 2006, 71, 8424.
Stolow, R. D.; Groom, T. Tetrahedron Lett. 1968, 9, 5781.
Standard Rational for Stereochemistry
O
H
FlaO
OH
O
O
R S
R L
4-hydroxyl cyclohexanone
44 56
FlaO
O
O
O
H
O
O
H
OH
H
Serine 432

O
Me
O
OAc
New Approach to Baeyer-Villiger CASTing
Clouthier, C. M.; Kayser, M. M.; Reetz, M. T. J. Org. Chem. 2006, 71, 8431.
O
R
O 2
CPMO
CHMO CPMO
143 + 432 156 + 450
WT GlyPhe-SerTyr GlyPhe-GlyIle GlyPhe-GlyCys
100 (46R)
27 (65R) 74 (92R) 37 (68R)
81 (5S) 20 (59R) 100 (8R) 89 (13S)
R
O
O
(S)

O
Me
O
OAc
New Approach to Baeyer-Villiger CASTing
Clouthier, C. M.; Kayser, M. M.; Reetz, M. T. J. Org. Chem. 2006, 71, 8431.
O
R
O 2
CPMO
CHMO CPMO
143 + 432 156 + 450
WT PheGly-LeuPhe PheGly-AsnTyr PheGly-HisLeu
100 (46R)
89 (91R) 67 (88R) 69 (80R)
81 (5S) 10 (ND) 19 (90R) 55 (74R)
R
O
O
(S)

Me
Mutant
Me
Oxidation of Thioethers
S Me
O 2, CHMO
Reetz, M. T.; Daligault, F.; Brunner, B.; Hinrichs, H.; Deege, A. Angew. Chem. Int. Ed. 2004, 43, 4078.
Me
Me
O O
S Me
S Me
Amino Acid
Exchanges Yield (%) Configuration ee% Sulfone (%)
WT - 75 R
14

In Cells:
O
N
(S,S) 82% ee, 96% de
(S,S) 79% ee, 96% de
Using Purified Enyzmes:
O
N
OH
OH
(S,S) 87% ee, 96% de, 90% yield
(S,S) 86% ee, 96% de, 85% yield
Cytochrome P450
O
B, 139-3 1-12G
Münzer, D. F.; Meinhold, P.; Peters, M. W.; Feichtenhofer, S.; Griengl, H.; Arnold, F. H. Chem. Commun. 2005, 2597.
Background:
Farinas, E. T.; Schwaneberg, U.; Glieder, A.; Arnold. F. H. Adv. Synth. Catal. 2001, 343, 601.
Glieder, A.; Farinas, E. T.; Arnold, F. H. Nat. Biotechnol. 2002, 20, 1135.
Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold. F. H. J. Am. Chem. Soc. 2003, 125, 13442.
N
Low Conversion 1-15%
O
B, 139-3 1-12G
N
O
N
OH
(R,R) 89% ee, 94% de
O
N
OH
(R,R) 89% ee, 95% de, 97% yield

H
N
O OH
Fe II
N N
S
N
O
Fe V
N N
N
S
H
N
H N
O OH
Fe II
N N
S
N
N
Cytochrome P450
N N
Meunier, B.; de Visser, S. E.; Shaik, S. Chem. Rev. 2004, 104, 3947.
H
N
O O
Fe II
N N
S
N
O
Fe V
S
OH
H
Fe IV
N N
N
S
N
N
H
N
Fe II
N N
S
N
N
H
N N
N
Fe III
N N
Fe III
S
S
N
OH
N
N
Fe III
N N
S
H
N

- Quick-E-Test
C 8H 17
C 13H 27
Me
O
O
O
O
- pH Indicator
C 8H 17
NO 2
O O
N
Me
O
O R
HO NO 2
H 2O
Lipase Mutants
H 2O, Buffer
Lipase
Screening
C 8H 17
Me
C 13H 27
C 8H 17
O
O
Me
OH
O
O
O
C 8H 17
Me
O
O
O N
NO 2
O O
UV Absorptions:
570 nm
C 8H 17
Me
O
O NO 2
UV Absorptions:
OH
410 nm

C 5H 11
Me
O
O
Lipase of a Different Class
NO 2
H 2O
Lipase Mutants
Sandström, A. G.; Engström, K.; Nyhlén, J.; Kasrayan, A.; Bäckvall, J.-E. Protein Eng., Des. Sel. 2009, 22, 413.
C 5H 11
Me
O
OH
C 5H 11
Me
Phe233Asn
Gly237Leu
Phe233Asn
Gly237Leu
Phe149Ser
Lle150Asp
O
O
WT
NO 2
S - 19 R - 27
O
NO 2
S - 52 No Improvement
Phe233Leu
Gly237Tyr
Asp95 is essential for stabilization of oxyanion
tetrahedral intermediate

RO
Phe233Gly
Best Mutant = Phe149Tyr
Lle150Asp
S - Selective Variant
RO
O
O
Et
Me
E = 84
Ph
RO
MeO
O
O
Me
Selectivity
Me
NonylO
Sandström, A. G.; Engström, K.; Nyhlén, J.; Kasrayan, A.; Bäckvall, J.-E. Protein Eng., Des. Sel. 2009, 22, 413.
Engström, K.; Nyhlén, J.; Sandström, A. G.; Bäckvall, J.-E. J. Am. Chem. Soc. 2010, 132, 7038.
PhO
O
O
Me
Me
E = 79 E = 276 E = 650
E = 211
E = 657
R - Selective Variant

Me
O
P. fluorescens
Esterase
Horsman, G. P.; Liu, A. M. F.; H, H.; Bornscheuer, U. T.; Kazlauskas, R. J. Chem. Eur. J. 2003, 9, 1933.
Schmidt, M.; Hasenpusch, D.; Kähler, M.; Kirchner, U.; Wiggenhorn, K.; Langel, W.; Bornscheuer, U. T.
ChemBioChem. 2006, 7, 805. Bornscheuer, U. T.; Altenbuchner, J.; Meyer, H. H. Biotechnol. Bioeng. 1998,
58, 544. Henke, E.; Bornscheuer, U. T. Biol. Chem. 1999, 380, 1029.
O
P. fluorescens
Ph OEt
Me O
Br OEt
Me
Ph OH
Mutations near active site
WT E = 3.8
Mutant E = 12
Mutation far from active site
Ac
O
Me
O
Ph OEt
P. fluorescens OH OAc
WT E = 3
2 mutation far from active site E = 96
1 mutation near to active site E = 90
Mutation close AND distal can have a significant affect on enantioselectivity
O
Br OEt
Me
O
Br OH
Me
WT E = 12
Mutant E = 19

Cy
•
Me
O
O
NO 2
Mutations for not necessarily smaller
Expanding to Axial Chirality
H 2O
Lipase Mutants
Carballeira, J. D.; Krumlinde, P.; Bocola, M.; Vogel, A.; Reetz, M. T.; Bäckvall, J.-E. Chem. Commun. 2007, 1913.
Cy
•
Me
O
OH
Cy
WT E = 8.6
Variant
Leu162Phe
Leu162Val
Leu162Ile
Leu162Ala
Leu162Thr
•
Me
O
O
NO 2
Conversion (%)
44
40
39
29
23
O NO 2
E-Value
111
39
33
13
10

N
N
N N N
Replacing Phe with Ala opens
active site to more diverse
substrates
Cytochrome P450
O
O
R
R = Me, Et, n-Pr, n-Bu
O
O
P450 Enzyme
C450 Mutant
Landwehr, M.; Hochrein, L.; Otey, C. R.; Alex Kasrayan, A.; Bäckvall, J.-E.; Arnold, F. H. J. Am. Chem. Soc. 2006, 128, 6058
Background:
Farinas, E. T.; Schwaneberg, U.; Glieder, A.; Arnold. F. H. Adv. Synth. Catal. 2001, 343, 601.
Glieder, A.; Farinas, E. T.; Arnold, F. H. Nat. Biotechnol. 2002, 20, 1135.
Peters, M. W.; Meinhold, P.; Glieder, A.; Arnold. F. H. J. Am. Chem. Soc. 2003, 125, 13442.
N
N
OH
O
O
R
O
N N N
9-10A-F87A
Et 57% ee
Pr 89% ee
Bu 94% ee
O
72% yield, 99% ee
OH

TTT Phenylalanine
TTC Phenylalanine
TTA Leucine
TTG Leucine
CTT Leucine
CTC Leucine
CTA Leucine
CTG Leucine
ATT Isoleucine
ATC Isoleucine
ATA Isoleucine
ATG Methionine
GTT Valine
GTC Valine
GTA Valine
GTG Valine
Kayser, M. M.; Clouthier, C. M. J. Org. Chem. 2006, 71, 8424.
Stolow, R. D.; Groom, T. Tetrahedron Lett. 1968, 9, 5781.
Codon Degeneracy - NDK
TCT Serine
TCC Serine
TCA Serine
TCG Serine
CCT Proline
CCC Proline
CCA Proline
CCG Proline
ACT Threonine
ACC Threonine
ACA Threonine
ACG Threonine
GCT Alanine
GCC Alanine
GCA Alanine
GCG Alanine
TAT Tyrosine
TAC Tyrosine
TAA Stop
TAG Stop
CAT Histidine
CAC Histidine
CAA Glutamine
CAG Glutamine
AAT Asparagine
AAC Asparagine
AAA Lysine
AAG Lysine
GAT Aspartic Acid
GAC Aspartic Acid
GAA Glutamic Acid
GAG Glutamic Acid
TGT Cysteine
TGC Cysteine
TGA Stop
TGG Tryptophan
CGT Arginine
CGC Arginine
CGA Arginine
CGG Arginine
AGT Serine
AGC Serine
AGA Arginine
AGG Arginine
GGT Gylcine
GGC Gylcine
GGA Gylcine
GGG Gylcine

TTT Phenylalanine
TTC Phenylalanine
TTA Leucine
TTG Leucine
CTC Leucine
CTA Leucine
ATT Isoleucine
ATC Isoleucine
ATA Isoleucine
GTT Valine
GTC Valine
GTA Valine
Kayser, M. M.; Clouthier, C. M. J. Org. Chem. 2006, 71, 8424.
Stolow, R. D.; Groom, T. Tetrahedron Lett. 1968, 9, 5781.
Codon Degeneracy - DNT
TCT Serine
TCC Serine
TCA Serine
CCC Proline
CCA Proline
ACT Threonine
ACC Threonine
ACA Threonine
GCT Alanine
GCC Alanine
GCA Alanine
TAT Tyrosine
TAC Tyrosine
TAA Stop
CAC Histidine
CAA Glutamine
AAT Asparagine
AAC Asparagine
AAA Lysine
GAT Aspartic Acid
GAC Aspartic Acid
GAA Glutamic Acid
TGT Cysteine
TGC Cysteine
TGA Stop
CTT Leucine CCT Proline CAT Histidine CGT Arginine
CTG Leucine
ATG Methionine
GTG Valine
TCG Serine
CCG Proline
ACG Threonine
GCG Alanine
TAG Stop
CAG Glutamine
AAG Lysine
GAG Glutamic Acid
TGG Tryptophan
CGC Arginine
CGA Arginine
CGG Arginine
AGT Serine
AGC Serine
AGA Arginine
AGG Arginine
GGT Gylcine
GGC Gylcine
GGA Gylcine
GGG Gylcine