Divergent roles of active-site and access-channel residues in controlling regioselectivity and catalytic efficiency in CYP154C10.

Cytochrome P450 (CYPs) catalyze regioselective oxidation reactions, yet the structural determinants governing regioselectivity and catalytic efficiency remain poorly understood. Here, we characterize CYP154C10 as a versatile steroid hydroxylase exhibiting predominant C2 regioselectivity and expanded substrate scope relative to previously described CYP154C enzymes. Notably, CYP154C10 supports steroid conversion using both chemical (PIDA) and NADPH-dependent (Fdx/FdR) systems, while maintaining consistent product distribution across both oxidation systems. Structure-guided mutagenesis, kinetic analysis, and molecular dynamics (MD) simulations together provide mechanistic insights into the functional separation between active-site and access-channel residues. Mutation of the active-site residue L93 to phenylalanine (L93F) induces substrate-dependent changes in regioselectivity by increasing conformational flexibility and enabling multiple catalytically competent substrate orientations. In contrast, mutations at the access-channel position (M199F/Y) preserve regioselectivity but modulate catalytic efficiency by perturbing substrate positioning and local hydration dynamics. Comparative structural and dynamic analyses further suggest that active-site architecture governs substrate orientation, whereas access-channel residues influence substrate delivery and catalytic efficiency in CYP154C10. Together, these findings provide a mechanistic framework for the rational engineering of selective P450 biocatalysts with tunable regioselectivity and catalytic efficiency.