Altering the Regioselectivity of Cytochrome P450 BM3 Variant M13 toward Genistein through Protein Engineering and Variation of Reaction Conditions

[Image: see text] The biocatalysts responsible for the enzymatic synthesis of hydroxygenisteins, derivatives of genistein with multiple activities, usually show regioselective promiscuity, hydroxylating genistein to form a mixture of multiple products, which, in turn, results in a cumbersome separation and purification. Hence, it is highly desired to explore the underlying mechanism regulating the regioselectivity of hydroxylases. M13 is a variant of cytochrome P450 BM3 with oxidant activity toward genistein. Herein, genistein was demonstrated to be hydroxylated by M13 to form a mixture of 3′-hydroxygenistein (3′-OHG) and 8-hydroxygenistein (8-OHG), each giving 4% conversion with a ratio of 1:1. Protein engineering toward M13 was thus performed to improve its regioselectivity. When isoleucine at position 86 was mutated into cysteine, the resultant variant M13I86C displayed improved regioselectivity toward 3′-OHG with an increased conversion of 8.5%. The double mutation M13I86CP18W further boosted the conversion of 3′-OHG to 9.6%, and the ratio of 3′-OHG to 8-OHG increased to 12:1. Conversely, both CoCl(2) and glucose 6-phosphate (G6P) could lead to more 8-OHG. When Co(2+) reached 37.5 mM, M13I86CP18W could give an 8-OHG conversion of 22.4%. The maximal ratio of 8-OHG to 3′-OHG reached 130 when 62.5 mM Co(2+) was included in the reaction mixture. With the increase of G6P from 10 to 40 mM, the conversion of M13I86CP18W to 8-OHG gradually increased to 22.6%, while the conversion to 3′-OHG decreased to 6%. Thus, both intrinsic residues and external reaction conditions can affect the regiospecificity of M13, which laid the foundation for the selection of suitable biocatalysts for the hydroxylation of genistein.