Role of Residue 87 in Substrate- and Regioselectivity of Drug Metabolizing Cytochrome P450 Bm3 M11

BM3, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed a drug metabolizing mutant BM3 M11 by a combination of site-directed and random mutagenesis. BM3 M11 contains ten mutations, when compared to wild-type BM3 and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug metabolizing mutant BM3 M11 was mutated to all possible natural amino acids in order to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate-selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants extremely fast rates of NADPH-oxidation were observed (up to 6000 min -1 ). However, the coupling efficiencies were extremely low: even for the substrates showing highest rates of Odealkylation, coupling efficiencies were lower than 1%. With testosterone as substrate, all mutants were able to produce three hydroxytestosterone metabolites although at different activities and with remarkably different product ratios. The results show that the nature of amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant BM3 M11. Because of the wide substrate selectivity of BM3 M11 when compared to wild-type BM3, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.