Functional characterization of steroid hydroxylation by cytochrome P450 enzymes from Rhodococcus erythropolis KB1

Cytochrome P450 monooxygenases (CYPs) constitute a versatile family of biocatalysts capable of regio- and stereoselective hydroxylation, offering significant potential for pharmaceutical synthesis and the environmental remediation of endocrine-disrupting steroids. Although their synthetic utility is widely recognized, the mechanistic basis of their activity, particularly in bacteria, remains insufficiently explored. Building on prior discovery of steroid-degrading activity in Rhodococcus erythropolis KB1, in this study, we functionally characterized three putative P450 enzymes (CYP125ARh1, CYP125ARh2, and CYP153ARh3) identified from its genome. Substrate-binding assays demonstrated strong affinity toward progesterone and testosterone, but negligible affinity for 11β-hydroxysteroids, including cortisol and corticosterone. In vitro conversion assays revealed that all three enzymes specifically catalyze 16α-hydroxylation of progesterone and testosterone, although with varying catalytic efficiencies. Among them, CYP125ARh2 was identified as the most efficient enzyme, achieving near-quantitative conversion of progesterone (88%) and testosterone (62%). Kinetic analysis further underscored the superior catalytic properties of CYP125ARh2, which displayed lower Michaelis constants (Km = 16.41 ± 1.97 µM for progesterone and 24.50 ± 2.60 µM for testosterone) and higher turnover rates (Vmax = 6.05 ± 0.16 µM/min and 5.09 ± 0.13 µM/min, respectively). The superior catalytic efficiency of CYP125ARh2 originates from its unique active site architecture, which integrates a pre‑organized, synergistic residue network with pronounced induced‑fit plasticity. This structural synergy enables precise substrate positioning and optimal reaction geometry, thereby rationalizing its high 16α‑hydroxylation activity toward both progesterone and testosterone. These findings highlight CYP125ARh2 as a promising template for engineering high-performance biocatalysts. Overall, this work provides foundational insights into the role of the P450 enzymes in steroid chemistry and the biodegradation of steroidal pollutants.