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

Journal Article · · Angewandte Chemie
ORCiD logo [1];  [2];  [3]; ORCiD logo [4];  [3]; ORCiD logo [5]; ORCiD logo [6]
  1. College of Chemistry University of California, Berkeley Berkeley CA 94270 USA, Joint Bioenergy Institute (JBEI) Lawrence Berkeley National Laboratory Emeryville CA 94608 USA, Current address: Scripps Institution of Oceanography University of California, San Diego La Jolla CA 92037 USA
  2. Department of Energy Joint Genome Institute (DOE JGI) Lawrence Berkeley National Laboratory USA
  3. Joint Bioenergy Institute (JBEI) Lawrence Berkeley National Laboratory Emeryville CA 94608 USA
  4. Department of Energy Joint Genome Institute (DOE JGI) Lawrence Berkeley National Laboratory USA, Environmental Genomics and Systems Biology Lawrence Berkeley National Laboratory USA
  5. Joint Bioenergy Institute (JBEI) Lawrence Berkeley National Laboratory Emeryville CA 94608 USA, Department of Energy Joint Genome Institute (DOE JGI) Lawrence Berkeley National Laboratory USA, Environmental Genomics and Systems Biology Lawrence Berkeley National Laboratory USA
  6. College of Chemistry University of California, Berkeley Berkeley CA 94270 USA, Joint Bioenergy Institute (JBEI) Lawrence Berkeley National Laboratory Emeryville CA 94608 USA, Biological Systems and Engineering Division Lawrence Berkeley National Laboratory USA, Center for Biosustainability Danish Technical University Lyngby Denmark, Center for Synthetic Biochemistry Institute for Synthetic Biology Shenzhen Institutes of Advanced Technology Shenzhen 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.

Sponsoring Organization:
USDOE
OSTI ID:
1526087
Journal Information:
Angewandte Chemie, Journal Name: Angewandte Chemie Vol. 131 Journal Issue: 30; ISSN 0044-8249
Publisher:
Wiley Blackwell (John Wiley & Sons)Copyright Statement
Country of Publication:
Germany
Language:
English

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