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

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.

Authors:
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
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1545150
DOE Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article
Journal Name:
Angewandte Chemie (International Edition)
Additional Journal Information:
Journal Volume: 58; Journal Issue: 30; Journal ID: ISSN 1433-7851
Publisher:
Wiley
Country of Publication:
United States
Language:
English

Citation Formats

de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., and Keasling, Jay D. A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts. United States: N. p., 2019. Web. doi:10.1002/anie.201901782.
de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., & Keasling, Jay D. A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts. United States. doi:10.1002/anie.201901782.
de Rond, Tristan, Gao, Jian, Zargar, Amin, de Raad, Markus, Cunha, Jack, Northen, Trent R., and Keasling, Jay D. Thu . "A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts". United States. doi:10.1002/anie.201901782.
@article{osti_1545150,
title = {A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts},
author = {de Rond, Tristan and Gao, Jian and Zargar, Amin and de Raad, Markus and Cunha, Jack and Northen, Trent R. and Keasling, Jay D.},
abstractNote = {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 P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.},
doi = {10.1002/anie.201901782},
journal = {Angewandte Chemie (International Edition)},
issn = {1433-7851},
number = 30,
volume = 58,
place = {United States},
year = {2019},
month = {6}
}

Works referenced in this record:

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A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes
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A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes
journal, July 2002


Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3
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Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties
journal, November 2007

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