Screening for cytochrome p450 reactivity by harnessing catalase as reporter enzyme

Chembiochem. 2009 Mar 2;10(4):751-7. doi: 10.1002/cbic.200800750.

Abstract

Cytochrome P450 enzymes are known to catalyze a variety of reactions that are difficult to perform by standard organic synthesis, such as the oxidation of unactivated C--C bonds. Cytochrome P450 enzymes can also be used in artificial systems in which organic peroxides act as cosubstrates. To find substrates that are converted by a certain P450 catalyst in the presence of an organic peroxide, various screening assays have been established, however, most of them are limited to one or only a few specific substrates. Here, we report a simple and rapid screening assay that works independently of the nature of the substrate and utilizes a previously undescribed reactivity of catalase as reporter enzyme. In an initial demonstration of this assay, we screened 180 enzyme/peroxide/substrate combinations for potential bioconversions. As shown by subsequent verification of the screening results with liquid chromatography/multistage mass spectrometry (LC/MS(n)), we were able to identify three new substrates for the enzyme CYP152A1 and at least two previously undescribed conversions by the enzyme CYP119.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay / methods*
  • Calibration
  • Catalase / analysis
  • Catalase / metabolism*
  • Chromatography, Liquid
  • Cytochrome P-450 Enzyme System / analysis*
  • Cytochrome P-450 Enzyme System / chemistry
  • Cytochrome P-450 Enzyme System / metabolism*
  • Feasibility Studies
  • Mass Spectrometry
  • Models, Molecular
  • Peroxides / metabolism
  • Protein Conformation
  • Reproducibility of Results
  • Time Factors

Substances

  • Peroxides
  • Cytochrome P-450 Enzyme System
  • Catalase