Regioselectivity and Activity of Cytochrome P450 BM-3 and Mutant F87A in Reactions Driven by Hydrogen Peroxide

Abstract

Cytochrome P450 BM-3 (EC 1.14.14.1) is a monooxygenase that utilizes NADPH and dioxygen to hydroxylate fatty acids at subterminal positions. The enzyme is also capable of functioning as a peroxygenase in the same reaction, by utilizing hydrogen peroxide in place of the reductase do- main, cofactor and oxygen. As a starting point for developing a practically useful hydroxylation bio- catalyst, we compare the activity and regioselectivity of wild-type P450 BM-3 and its F87A mutant on various fatty acids. Neither enzyme catalyzes ter- minal hydroxylation under any of the conditions studied. While significantly enhancing peroxygenase activity, the F87A mutation also shifts hydroxylation further away from the terminal position. The H2O2- driven reactions with either the full-length BM-3 enzyme or the heme domain are slow, but yield product distributions very similar to those generated when using NADPH and O2.