Engineering P450 Monooxygenases for Highly Regioselective and Active p-Hydroxylation of m-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 (k cat/K M 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.

Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3

“…34,39,40 Introduction of mutations R47I and R47L in P450BM3 can increase hydrophobicity and expand the channel, thereby facilitating better access of m-alkylphenol to the heme center. 28 Consistent with the findings of this study, P25A-P329A-E435D of VD-BM3 and A328G-L437G-L439G of FA11a1-BM3 can create larger, more accommodating channel surfaces that facilitate the access of more steroids to the heme center. However, the substrate channels are often composed of numerous hydrophobic residues.…”

“…Efficient access of the substrate into the heme pocket is vital for the P450BM3 catalytic process. 28 The initial P450BM3 engineering strategy aimed to expand the substrate entry to facilitate access of PG to the heme center. It has been noted that the wild-type (WT)-P450BM3 cannot hydroxylate PG (Table 1).…”

Directed Evolution of the UDP-Glycosyltransferase UGTBL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

“…58 The distance was the longest in mutant KRW 1 W 2 (D M4 ), indicating that the change in distance caused by substrate rotation may have an important effect on regioselectivity. 59 This has also been observed in many protein engineering studies. Wu et al achieved modification of Lisoleucine dioxygenase regioselectivity by fine-tuning the substrate-binding pose to expand the distance between C4 or C5 of the substrate and Fe.…”

“…This distance was significantly shorter in the mutant SMWRW 1 W 2 (D M6 ) than in the WT, which may facilitate protonation and deprotonation during the enzymatic reaction . The distance was the longest in mutant KRW 1 W 2 (D M4 ), indicating that the change in distance caused by substrate rotation may have an important effect on regioselectivity . This has also been observed in many protein engineering studies.…”

Computer-Aided Tunnel Engineering: A Promising Strategy for Improving Lipase Applications in Esterification Reactions

“…Considering the special role of the tunnel, lipase biocatalysis and biotransformation can be greatly improved by reconstructing the tunnel from the molecular and catalytic characteristics of lipase. In the past few years, a number of publications have focused on the design of substrate tunnels and have yielded a number of enzymes with excellent performance. ,− Therefore, rational design of the lipase tunnel precisely regulates the transport rate of water and accelerates the progress of the enzymatic esterification reaction, thus improving the efficiency of the enzymatic reaction. This work will help to solve the key scientific problem of “substrates and products passage” in enzymatic esterification systems and provide ideas to break the bottleneck in the application of esterification in industry.…”