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Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation

Dick, Markus and Sarai, Nicholas S. and Martynowycz, Michael W. and Gonen, Tamir and Arnold, Frances H. (2019) Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation. Journal of the American Chemical Society . ISSN 0002-7863. (In Press)

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We previously engineered the tryptophan synthase β-subunit (TrpB), which catalyzes the condensation of L-serine and indole to L-tryptophan, to synthesize a range of noncanonical amino acids from L-serine and indole derivatives or other nucleophiles. Here we employ directed evolution to engineer TrpB to accept 3-substituted oxindoles and form C–C bonds leading to new quaternary stereocenters. Initially, the variants that could use 3-substituted oxindoles preferentially formed N–C bonds by attacking N₁ of the substrate. Protecting N₁ encouraged evolution towards C-alkylation, which persisted when protection was removed. Six generations of directed evolution resulted in TrpB Pf_(quat) with a 400-fold improvement in activity for alkylation of 3-substituted oxindoles and the ability to selectively form a new, all-carbon quaternary stereo-center at the γ-position of the amino acid products. The enzyme can also alkylate and form all-carbon quaternary stereocenters on structurally similar lactones and ketones, where it exhibits excellent regioselectivity for the tertiary carbon. The configurations of the γ-stereocenters of two of the products were determined via microcrystal electron diffraction (MicroED), and we report the MicroED structure of a small molecule obtained using the Falcon III direct electron detector. Highly thermostable and expressed at >500 mg/L E. coli culture, TrpB Pf_(quat) offers an efficient, sustainable, and selective platform for the construction of diverse noncanonical amino acids bearing all-carbon quaternary stereocenters.

Item Type:Article
Related URLs:
URLURL TypeDescription Paper
Sarai, Nicholas S.0000-0002-4655-0038
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2019 American Chemical Society. Publication Date: November 20, 2019. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) and by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM125887. The Gonen lab is supported by funds from the Howard Hughes Medical Institute. The authors thank David C. Miller and Nathaniel W. Goldberg for assistance with experiments. The authors thank David K. Romney, Patrick J. Almhjell, Susanne Bähr, Nicholas J. Porter, Professor Brian M. Stoltz (Caltech) and Sanel Suljic for helpful discussions. 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).
Funding AgencyGrant Number
Deutsche Forschungsgemeinschaft (DFG)UNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Subject Keywords:TrpB, noncanonical amino acid, asymmetric catalysis, quaternary carbon, oxindole, C–C bond formation, directed evolution
Record Number:CaltechAUTHORS:20191022-085105295
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Official Citation:Tailoring Tryptophan Synthase TrpB for Selective Quaternary Carbon Bond Formation. Markus Dick, Nicholas S. Sarai, Michael W. Martynowycz, Tamir Gonen, and Frances H. Arnold. Journal of the American Chemical Society. Just Accepted Manuscript. DOI: 10.1021/jacs.9b09864
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99383
Deposited By: Tony Diaz
Deposited On:22 Oct 2019 20:28
Last Modified:21 Nov 2019 21:31

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