SemirationallyEngineering an Efficient P450 Peroxygenasefor Regio- and Enantioselective Hydroxylation of Steroids

Enzymatic direct hydroxylation of unactivated C–H bonds in steroids provides a promising approach to enrich their structural and functional diversity, together with higher physiological and pharmacological activity. Here, we construct an efficient peroxide-driven P450 hydroxylase for the regio- and enantioselective hydroxylation of steroids. The NADH-dependent CYP154C5 monooxygenase is smoothly transformed into its peroxygenase mode by combining the strategies of H₂O₂ tunnel engineering and the introduction of a catalytic aspartate residue, which avoids the use of expensive nicotinamide cofactors and redox partner proteins. The variant F92A/R114A/E282A/T248D (AAA/T248D) quantitatively converted testosterone and nandrolone into the corresponding 16α-hydroxylation products, showing the best catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) for testosterone hydroxylation among all known natural and engineered P450 peroxygenases to date. Crystal structural analysis and molecular dynamics simulations suggest that H₂O₂ tunnel engineering plays a crucial role in promoting the flow of H₂O₂ into active centers, and the introduced aspartate residue may participate in the activation of H₂O₂. Moreover, the milligram-scale preparation of 16α-hydroxytestosterone by AAA/T248D gave a substrate conversion rate (>98%) and an isolated yield (90%), suggesting potential for synthetic application. This work not only establishes a feasible semirational approach to engineered non-natural P450 peroxygenases but also provides a potentially practical approach for the enzymatic synthesis of hydroxylated steroid compounds.