A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

de Rond, Tristan; Gao, Jian; Zargar, Amin; de Raad, Markus; Cunha, Jack; Northen, Trent R.; Keasling, Jay D.

doi:10.1002/anie.201901782

Title: A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

Journal Article · 29 May 2019 · Angewandte Chemie (International Edition)

DOI: https://doi.org/10.1002/anie.201901782 · OSTI ID:1545150

^[1]; Gao, Jian ^[2]; Zargar, Amin ^[3];

^[2]; Cunha, Jack ^[3];

^[4];

^[5]

University of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); University of California, San Diego, CA (United States). Scripps Institute of Oceanography
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Department of Energy Joint Genome Institute (DOE JGI)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). Department of Energy Joint Genome Institute (DOE JGI)
University of California, Berkeley, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Danmarks Tekniske University, Lyngby (Denmark); Shenzhen Institute of Advanced Technology, Shenzen (China)

Abstract Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450 _BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231; 1RC1GM090980; F32GM125179; F32GM129960; 1341894; 1442724

OSTI ID:: 1545150

Alternate ID(s):: OSTI ID: 1526088

Journal Information:: Angewandte Chemie (International Edition), Vol. 58, Issue 30; ISSN 1433-7851

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 20 works

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
high-throughput screening
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cytochrome P450

Title: A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

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