Electronic and steric factors in C–H bond hydroxylation of branched alkanes by cytochrome P450 enzymes

Branched alkanes, key components of crude oil, present significant challenges in bioconversion processes of the petroleum industry. In this study, we provide new insights into the hydroxylation of branched alkanes (C6–C8) catalyzed by CYP102A1 enzymes from Priestia megaterium. We engineered a series of CYP102A1 mutants and examined their catalytic properties through NADPH oxidation and GC-MS analysis. Notably, a quadruple mutant, R47L/F87V/L188Q/I259M, exhibited exceptional regioselectivity for tertiary and adjacent secondary C–H bonds. Computational calculation (docking and molecular dynamics simulation) revealed that substrate binding energy at the active site, rather than C–H bond dissociation energy alone, serves as the critical determinant of regioselectivity. Our study reveals that the positioning and rate of hydroxylation are not only determined by the C–H bond energy, but also by the structural complementarity between the substrate and enzyme.