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A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts
Author(s) -
de Rond Tristan,
Gao Jian,
Zargar Amin,
de Raad Markus,
Cunha Jack,
Northen Trent R.,
Keasling Jay D.
Publication year - 2019
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201901782
Subject(s) - chemistry , high throughput screening , enzyme , cytochrome p450 , substrate (aquarium) , cytochrome , enzyme assay , biocatalysis , drug discovery , chromatography , lysis , biochemistry , combinatorial chemistry , catalysis , biology , reaction mechanism , ecology
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.