Engineering P450 Monooxygenases for Highly Regioselective and Active p-Hydroxylation of m-AlkylphenolsClick to copy article linkArticle link copied!
- Ren-Jie LiRen-Jie LiDepartment of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, SingaporeSynthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, 28 Medical Drive, Singapore 117456, SingaporeMore by Ren-Jie Li
- Kaiyuan TianKaiyuan TianDepartment of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, SingaporeMore by Kaiyuan Tian
- Xirui LiXirui LiDepartment of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, SingaporeMore by Xirui Li
- Anand Raghavendra GaikaiwariAnand Raghavendra GaikaiwariDepartment of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, SingaporeMore by Anand Raghavendra Gaikaiwari
- Zhi Li*Zhi Li*Email: [email protected]Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, SingaporeSynthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore, 28 Medical Drive, Singapore 117456, SingaporeMore by Zhi Li
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.
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