Discovery of a novel CYP107E41 from Streptoalloteichus sp. KCCM40925 for advancing steroid hydroxylation catalysis

Abstract

Cytochrome P450 enzymes (CYPs) are versatile biocatalysts involved in the biosynthesis, activation, and detoxification of a wide range of endogenous and exogenous compounds. While the CYP107 family is primarily associated with macrolide antibiotic biosynthesis, some members, such as OleP (CYP107D1) and CYP107X1, have also been shown to catalyze steroid hydroxylation, albeit with limited substrate scope. In this study, we identified CYP107E41 from Streptoalloteichus sp. KCCM40925 as a novel member of the CYP107 family with selective hydroxylation activity toward both androstane and pregnane steroids. In-vitro screening using a panel of eleven steroids revealed predominant 6β- and 16α-hydroxylation, with androstane substrates exhibiting higher conversion efficiencies. Among the redox systems evaluated, diacetoxyiodobenzene provided the highest catalytic efficiency. Molecular docking identified two major binding orientations, C6–Fe and C16–Fe, with the C6–Fe configuration consistently scoring higher in CNN pose evaluations, suggesting it as the catalytically preferred binding mode. Molecular dynamics simulations further supported this, showing that the testosterone complex maintained a stable conformation with C6 positioned near the heme iron, consistent with selective mono-hydroxylation. In contrast, the nandrolone complex exhibited greater conformational flexibility, maintaining the proximity of both C6 and C7 to the heme, aligning with its broader hydroxylation profile. These findings establish CYP107E41 as a promising candidate for regioselective steroid hydroxylation and provide mechanistic insights to inform future protein engineering and redox system optimization.