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Journal of the American Chemical Society
PfTrpB and StTrpB. Studies are ongoing to elucidate the role
and generality of this mysterious mutation.
NMR spectra (PDF)
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AUTHOR INFORMATION
Impact on substrate specificity. It is often thought that
evolving an enzyme for activity with a particular substrate
ultimately imparts specificity toward that substrate as well.
This study, however, shows that directed evolution toward a
particularly challenging substrate can be an effective strategy
to improve activity for nonꢀnative substrates in general. Inꢀ
deed, the intermediate catalyst, Pf5G8, exhibited concomitant
increases in activity for all four nitroindoles (Table 1). While
this enzyme was rarely the optimal catalyst, as indicated by its
infrequent appearance in Table 3, it exhibited good activity for
most substrates (see Table S1). The additional mutations in the
most evolved enzyme, Pf2A6, appeared to lower the substrate
generality, but nonetheless improved activity for multiple subꢀ
strates, such as 7ꢀnitroindole, in addition to the test substrate,
ORCID David K. Romney: 0000ꢀ0003ꢀ0498ꢀ7597
ORCIDꢀJavier MurcianoꢀCalles: 0000ꢀ0002ꢀ8667ꢀ1651
Corresponding Author
*
Eꢀmail: frances@cheme.caltech.edu.
ORCIDꢀFrances H. Arnold: 0000ꢀ0002ꢀ4027ꢀ364X
Funding Sources
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This work was funded by the Jacobs Institute for Molecular Medꢀ
icine (Caltech) and the Gordon and Betty Moore Foundation
(through the Caltech Programmable Molecular Technology Initiaꢀ
tive). D.K.R. was supported by a Ruth Kirschstein NIH Postdocꢀ
toral Fellowship (F32GM117635) and J.M.ꢀC. was supported by a
fellowship from the Alfonso Martín Escudero Foundation.
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ꢀnitroindole. Furthermore, the mutations of Pf2A6 served as
the basis to generate all other catalysts in this study, demonꢀ
strating that although a specific catalyst may not be optimal
for all substrates, the mutations are activating in multiple conꢀ
texts.
Notes
The contents of this paper are the subject of a patent application
submitted by Caltech, and some authors are entitled to a royalty
on revenues arising from that patent.
Our approach of creating a small panel of general catalysts
likely succeeded because the conversion of most nonꢀnative
substrates was limited by a single enzymatic process, Ser hyꢀ
drolysis. Thus, directed evolution could select for mutations
that curtailed Ser hydrolysis independently of a specific subꢀ
strate. This is consistent with the results of Table 4, in which
the rate of Ser deamination decreases even in the absence of a
nucleophilic substrate. While these specific circumstances
may not apply to all enzyme engineering problems, we would
expect directed evolution to provide a general benefit where
side activities that limit enzyme performance can be targeted
independently.
ACKNOWLEDGMENT
The authors thank Dr. Sabine BrinkmannꢀChen for advice on
developing the screening assay, Dr. Andrew Buller for helpful
discussions and comments on the manuscript, and Elliot Mackrell
for assistance in screening the catalyst libraries.
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CONCLUSION
4
By evolving for activity with nitroindoles, we have develꢀ
oped a panel of TrpBꢀderived biocatalysts that exhibit good to
excellent activity with monoꢀ and disubstituted indoles. The
substrate scope includes indoles bearing electronꢀwithdrawing
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stubborn nucleophiles. These qualities make TrpB catalysis a
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ble synthetic building blocks.
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The results also demonstrate that the universally conserved
E104 residue is a key target for mutagenesis to improve activiꢀ
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starting point to adapt TrpB for the synthesis of new product
classes. More generally, this demonstrates how mutations at
residues that seem crucial can in fact be the most valuable
handles for tuning activity with new substrates.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the
ACS Publications website.
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Additional figures and experimental procedures (PDF)
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