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Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

preprint
submitted on 11.09.2019 and posted on 12.09.2019 by Markus Dick, Nicholas S. Sarai, Michael W. Martynowycz, Tamir Gonen, Frances H. Arnold

We previously engineered the tryptophan synthase beta-subunit (TrpB), which catalyzes the condensation reaction between L-serine and indole to form L-tryptophan, to synthesize a range of modified tryptophans from serine and indole derivatives. In this study, we used directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. At first, the TrpBs that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking the oxindole N1 atom. We found, however, that protecting the nitrogen encouraged evolution towards C-alkylation, which persisted even when this protection was removed. After seven rounds of evolution leading to a 400-fold improvement in activity, variant Pfquat efficiently alkylates 3-substituted oxindoles to selectively form new stereocenters at the γ-position of the amino acid products. The configuration of the new γ-stereocenter of one of the products was determined from the crystal structure obtained by microcrystal electron diffraction (MicroED). Substrates structurally related to 3-methyloxindole such as lactones and ketones can also be used by the enzyme for quaternary carbon bond formation, where the biocatalyst exhibits excellent regioselectivity for the tertiary carbon atom. Highly thermostable and expressed at > 500 mg/L E. coli culture, TrpB Pfquat provides an efficient and environmentally-friendly platform for the preparation of noncanonical amino acids bearing quaternary carbons.

Funding

Deutsche Forschungsgemeinschaft (DFG)

National Institute of General Medical Sciences of the National Institutes of Health Award Number R01GM125887

Howard Hughes Medical Institute

History

Email Address of Submitting Author

frances@cheme.caltech.edu

Institution

California Institute of Technology

Country

United States

ORCID For Submitting Author

0000-0002-4027-364X

Declaration of Conflict of Interest

The authors declare the following competing financial interest: The contents of this paper are the subject of a patent application submitted by Caltech (M.D. inventor).

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