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/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.
Rabe
Kersten S
KS
Technische Universität Dortmund, Fakultät Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Strasse 6, Dortmund, Germany.
Spengler
Mark
M
Erkelenz
Michael
M
Müller
Joachim
J
Gandubert
Valerie J
VJ
Hayen
Heiko
H
Niemeyer
Christof M
CM
D001681:Biological Assay / Q000379:methods*; D002138:Calibration; D002374:Catalase / Q000032:analysis / Q000378:metabolism*; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D005240:Feasibility Studies; D013058:Mass Spectrometry; D008958:Models, Molecular; D010545:Peroxides / Q000378:metabolism; D011487:Protein Conformation; D015203:Reproducibility of Results; D013997:Time Factors
D010545:Peroxides; D003577:Cytochrome P-450 Enzyme System; D002374:Catalase