CYP102A1 peroxygenase catalyzed reaction via in situ HO generation.

CYP102A1 is a promiscuous bacterial cytochrome P450 (CYP or P450) known for its diverse substrates and comparable activity with human P450 enzymes. The development of CYP102A1 peroxygenase activity can contribute significantly to human drug development and drug metabolite production. Peroxygenase has recently emerged as an alternative to a dependency of P450 on NADPH-P450 reductase and NADPH cofactor and gives more opportunity for practical application. However, the HO dependency also leads to challenges regarding its practical application, in which the excessive HO concentration causes the activation of the peroxygenases. Therefore, we need the optimization of HO production to minimize oxidative inactivation. In this study, we report the CYP102A1 peroxygenase-catalyzed atorvastatin hydroxylation reaction with an enzymatic HO generation using glucose oxidase. Random mutagenesis at the CYP102A1 heme domain was used to generate mutant libraries with high throughput screening of highly active mutants, which can pair with the in situ HO generation. The setup of the CYP102A1 peroxygenase reaction was also possible for other statin drugs and could be developed to produce drug metabolites. We also found a relationship between enzyme inactivation and product formation during the catalytic reaction, supported by enzymatic in situ HO supply. It can be suggested that the low product formation is due to enzyme inactivation.