A High-Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts.
Tristan de RondJian GaoAmin ZargarMarkus de RaadJack CunhaTrent R NorthenJay D KeaslingPublished in: Angewandte Chemie (International ed. in English) (2019)
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.
Keyphrases
- high throughput
- single cell
- hydrogen peroxide
- mass spectrometry
- cell therapy
- high speed
- bone mineral density
- molecular dynamics simulations
- small molecule
- liquid chromatography
- label free
- real time pcr
- postmenopausal women
- high resolution
- simultaneous determination
- high performance liquid chromatography
- sensitive detection