Screening for Cytochrome P450 Reactivity by Harnessing Catalase as Reporter Enzyme

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/MSn), we were able to identify three new substrates for the enzyme CYP152A1 and at least two previously undescribed conversions by the enzyme CYP119.