Impacts of amino acid substitutions in fungal cytochrome P450 monooxygenase (CYP57B3) on the effective production of 3΄-hydroxygenistein

Aspergillus oryzae cytochrome P450 monooxygenase (CYP57B3) is capable of catalyzing hydroxylation of genistein to produce 3΄-hydroxygenistein. Because hydroxylated derivatives of genistein, including 3΄-hydroxygenistein, exhibit various pharmacological activities, CYP57B3 would potentially be useful as a biocatalyst in the pharmaceutical field. We therefore performed random mutagenesis of CYP57B3 to improve its catalytic activities for genistein. Random mutations were introduced by error-prone PCR into CYP57B3, resulting in construction of a library of mutants. From 2000 mutants, we isolated one with a triple mutation in CYP57B3, resulting in three amino acid substitutions (V138I, S243N and V463F). The altered protein, designated CYP57B3-mut, had a high level of activity for the production of 3΄-hydroxygenistein from genistein. In bioconversion reactions, Saccharomyces cerevisiae expressing CYP57B3-mut showed 14-fold higher production of 3΄-hydroxygenistein than that of wild-type CYP57B3. In vitro kinetic analysis revealed that Km values of wild-type and CYP57B3-mut were 14.6 × 103 ± 2.7 × 103 μM and 15.7 ± 1.0 μM, respectively. These results suggest that the affinity of CYP57B3 for genistein could be dramatically improved by the alterations of V138, S243 and V463 in CYP57B3.