Engineering P450 Monooxygenases for Highly Regioselective and Active <i>p</i>-Hydroxylation of <i>m</i>-Alkylphenols

Abstract

Regioselective hydroxylations of aromatic compounds are useful reactions but often lack appropriate catalysts. Here a group of P450BM3 mutants (R47I/A82F/A328F, R47L/Y51F/F87V/L188P/I401P, R47I/Y51F/F87V, R47L/Y51F/F87V/L181Q/L188P/I401P, and R47I/F87V/L188P) were developed as unique catalysts for the p-hydroxylation of m-alkylphenols 1a–e with high regioselectivity (91–99%) and conversion (95–99%) to produce the corresponding useful and valuable m-alkylbenzene-1,4-diols 2a–e, respectively. The mutated hydroxylases were developed by protein engineering of P450BM3 monooxygenase via site-directed mutagenesis based on designed mutations to reshape the substrate binding pocket and access channel. Several engineered P450BM3 mutants showed good catalytic efficiency (kcat/KM of 234–381 mM–1 min–1) for the p-hydroxylations of m-alkylphenols 1a–e, respectively. Molecular docking and simulation gave some insights into the structure-based understanding of the enhanced regioselectivity and activity for the developed P450BM3 mutants, including the shorter distance between heme-oxygen atom and C4-carbon (p-position) of substrates than the wild-type enzyme in the catalytic pockets. Preparative biohydroxylations of m-alkylphenols 1a–e were demonstrated by using E. coli cells coexpressing individual P450BM3 mutants and glucose dehydrogenase GDH, giving high-yielding synthesis of useful and valuable m-alkylbenzene-1,4-diols 2a–e.