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

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

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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 P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.
Original languageEnglish
JournalANGEWANDTE CHEMIE
Volume58
Issue number30
Pages (from-to)10114-10119
Number of pages7
ISSN1433-7851
DOIs
Publication statusPublished - 2019

Keywords

  • Biocatalysis
  • Cytochrome P450
  • Enzyme assays
  • High-throughput screening
  • Mass spectrometry

Cite this

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. In: ANGEWANDTE CHEMIE. 2019 ; Vol. 58, No. 30. pp. 10114-10119.
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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 P450BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.",
keywords = "Biocatalysis, Cytochrome P450, Enzyme assays, High-throughput screening, Mass spectrometry",
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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.

In: ANGEWANDTE CHEMIE, Vol. 58, No. 30, 2019, p. 10114-10119.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

AU - de Rond, Tristan

AU - Gao, Jian

AU - Zargar, Amin

AU - de Raad, Markus

AU - Cunha, Jack

AU - Northen, Trent R.

AU - Keasling, Jay D.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Biocatalysis

KW - Cytochrome P450

KW - Enzyme assays

KW - High-throughput screening

KW - Mass spectrometry

U2 - 10.1002/anie.201901782

DO - 10.1002/anie.201901782

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IS - 30

ER -