H2O2-driven hydroxylation of steroids catalyzed by cytochrome P450 CYP105D18: Exploration of substrate access channel

Abstract

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well, and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a co-substrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at the position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (Km/kcat) towards testosterone was increased 1.36-fold in F184A mutant as compared with the wild type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications.