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.
Site-Selective C-H Halogenation using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling
20 August 2019, Version 1
This content is a preprint and has not undergone peer review at the time of posting.
FDH genome mining SI