A high-throughput mass spectrometric enzyme activity assay enabling the discovery of cytochrome P450 biocatalysts

TitleA high-throughput mass spectrometric enzyme activity assay enabling the discovery of cytochrome P450 biocatalysts
Publication TypeJournal Article
Year of Publication2019
Authorsde Rond T., Gao J., Zargar A., de Raad M., Cunha J., Northen T.R, Keasling J.D
Volume58
Pagination10114-10119
Date Published2019/07
Type of ArticleArticle
ISBN Number1433-7851
Accession NumberWOS:000476452700008
Keywordsbiocatalysis; chemistry; cytochrome P450; directed evolution; enantioselectivity; enzyme assays; high-throughput screening; hydroxylation; identification; mass spectrometry; monooxygenases; nootkatone; regio
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.

DOI10.1002/anie.201901782
Student Publication: 
No
Research Topics: 
sharknado