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Screening for Cytochrome P450 Reactivity by Harnessing Catalase as Reporter Enzyme
Author(s) -
Rabe Kersten S.,
Spengler Mark,
Erkelenz Michael,
Müller Joachim,
Gandubert Valerie J.,
Hayen Heiko,
Niemeyer Christof M.
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800750
Subject(s) - catalase , chemistry , substrate (aquarium) , cytochrome p450 , enzyme , peroxide , peroxidase , reactivity (psychology) , combinatorial chemistry , mass spectrometry , biochemistry , organic synthesis , high throughput screening , cytochrome , chromatography , catalysis , organic chemistry , biology , medicine , ecology , alternative medicine , pathology
The big screen : We have devised a high‐throughput screening method for organic peroxide‐dependent P450 reactivity by taking advantage of a previously undescribed activity of catalase, which was used as reporter enzyme. This two‐step assay, followed by liquid chromatography/mass spectrometry analyses, allowed the facile identification of several new substrates for bacterial P450 enzymes.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 CC 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.