A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts. (13th June 2019)

• Record Type: Journal Article
• Authors: de Rond, Tristan; Gao, Jian; Zargar, Amin; de Raad, Markus; Cunha, Jack; Northen, Trent R.; Keasling, Jay D.
• Is Part Of: Angewandte Chemie. Volume 131:Number 30(2019)
• Page Start: 10220
• Page End: 10225
• Publication Date: 2019-06-13
• Subjects: Biokatalyse -- Cytochrom P450 -- Enzym-Assays -- Hochdurchsatz-Screening -- Massenspektrometrie; Chemistry -- Periodicals; 540
• DOI: 10.1002/ange.201901782
• Languages: English
• ISSNs: 0044-8249

Abstract

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. Abstract : Ein Hochdurchsatz‐Enzymassay wurde entwickelt, der auf einer Kombination aus Klickchemie, Fluoraffinitätsreinigung und Nanostrukturinitiator‐Massenspektrometrie basiert. Der Nutzen dieser PECAN‐Technik („probing enzymes with click‐assisted NIMS") wurde durch das Screening einer Bibliothek von Cytochrom‐P450‐Enzymen auf Aktivität in der Valencen‐Oxidation demonstriert.