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Site-Selective C-H Halogenation using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling

preprint
submitted on 19.08.2019 and posted on 20.08.2019 by Brian F. Fisher, Harrison M. Snodgrass, Krysten A. Jones, Mary C. Andorfer, Jared C. Lewis

Herein, we describe the use of a high-throughput mass spectrometry-based screen to evaluate a broad set of over one hundred putative FDH sequences drawn from throughout the FDH family. Halogenases with novel substrate scope and complementary regioselectivity on large, three-dimensionally complex compounds were identified. This effort involved far more extensive sequence-function analysis than has been accomplished using the relatively narrow range of FDHs characterized to date, providing a clearer picture of the regions in FDH sequence space that are most likely to contain enzymes suitable for halogenating small molecule substrates. The representative enzyme panel constructed in this study also provides a rapid means to identify FDHs for lead diversification via late-stage C-H functionalization. In many cases, these enzymes provide activities that required several rounds of directed evolution to accomplish in previous efforts, highlighting that this approach can achieve significant time savings for biocatalyst identification and provide advanced starting points for further evolution.

Funding

NIH (1R01GM115665)

NSF (CCHF, CHE-1700982)

NIH (F32GM123693)

DOE (DE-AC02-05CH11231)

History

Email Address of Submitting Author

jcl3@iu.edu

Institution

Indiana University

Country

USA

ORCID For Submitting Author

0000-0003-2800-8330

Declaration of Conflict of Interest

no conflict of interest

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