Abstract
Steroids are among the most valuable and widely used pharmaceuticals. The cholesterol side-chain cleavage enzyme (P450scc) is critical for steroid metabolism and hormone biosynthesis. While mammalian eukaryotic P450scc enzymes are well-characterized, bacterial counterparts remain underexplored despite their industrial promise and potential contributions to bacterial steroid catabolism. Here, we identify a series of CYP204 family P450 enzymes, widely distributed across diverse steroid-degrading bacterial species, that catalyze the side-chain cleavage of cholesterol, phytosterol, and cholestenone to produce pregnenolone and progesterone. Unlike mammalian enzymes, which exhibit strict cholesterol specificity, bacterial P450scc enzymes display relaxed substrate specificity, preferentially converting cholestenone to progesterone-a key precursor in steroid drug semi-synthesis. Structural and mechanistic analyses demonstrate that CYP204 enzymes employ a flexible, dual-regioselective C-H activation mechanism distinct from the sequential hydroxylation of mammalian P450scc enzymes. Iterative saturation mutagenesis identified critical residues for side-chain cleavage, improving catalytic efficiency up to 6.5-fold, and computational analyses clarified sequence-function relationships. This finding of bacterial P450scc enzymes not only underscores their potential function in bacterial steroid catabolism but also lays a foundation for promising biocatalytic strategies for pregnenolone and progesterone synthesis.