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7.344 Directed Evolution: Engineering Biocatalysts
Spring 2008
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Enzyme evolution by genetic
complementation
Yano, T.; Oue, S.; Kagamiyama, H. Directed evolution of an
aspartate aminotransferase with new substrate specificities.
Proc. Natl. Acad. Sci. USA 1998, 95, 5511-5515.
Otten, L.G.; Sio, C.F.; Vrielink, J.; Cool, R.H.; Quax, W.J.
Altering the substrate specificity of cephalosporin acylase by
directed evolution of the β-subunit. J. Biol. Chem. 2002, 277
(44), 42121-42127.
Evolution of an aspartate
aminotransferase
• What are the authors trying to do?
• What is the process by which they conduct their
evolution? Explain the Materials and Methods
section.
• What method of library generation do they use?
Is this a good choice?
• How do they carry out their selection?
• What are the results? Does their explanation of
why mutants lead to increased activity make
sense?
• Why is this evolution worthwhile? JOC paper
Aminotransferase catalysis
Image of aminotransferase catalytic cycle removed
due to copyright restrictions.
Explanation of mutations from
crystal data
Image removed due to copyright restrictions.
Please see Fig. 3 in Yano, T., S. Oue, and H.
Kagamiyama. “Directed evolution of an aspartate
aminotransferase with new substrate specificities.”
PNAS. 95(1998): 5511-5515.
Evolution of a cephalosporin
acylase
• What are the authors trying to do? Why is
this significant?
• How do the authors make their library? Is
this reasonable?
• What is the selection method in this
evolution?
• What are the results?
• What are the problems or pitfalls in this
paper?
Cephalosporin synthetic pathway
H
N
H 2N
S
O
CO2H
CH3
N
CH3
O
CO2H
Adipate feed
Acyltransferase
H
N
S
O
CO2H
N
CH3
CH3
O
CO2H
Expandase/
Hydroxylase
Expandase
H
N
O
CO2H
H
N
S
N
O
CO2H
O
S
N
O
CH3
CH3OH
CO2H
CO2H
Acetyltransferase
Acylase
H
N
S
O
CO2H
O
N
O
CH2OCCH3
CO2H
H2N
S
Acylase
O
N
O
CH2OCCH3
H 2N
S
CO2H
O
N
O
CH2OCCH3
CO2H
Positions of mutated residues
Image removed due to copyright restrictions.
Please see Fig. 4 in Otten, L.G., C. F. Sio, J.
Vrielink, R. H. Cool, and W. J. Quax. “Altering the
substrate specificity of cephalosporin acylase by
directed evolution of the β-subunit.” J. Biol. Chem.
277(2002): 42121-42127.
For next week…chemical
complementation
Transcriptional
Activator
E
SUBSTRATE
DNA binding
domain
reporter gene