Rational Design of a Cytochrome P450 for Efficient 11α-Hydroxylation of Steroids by Integrating Genome Mining and Global Dynamic Simulations

Abstract The 11α-hydroxylation of steroids is essential for their semisynthesis. However, natural 11α-hydroxylases are scarce and generally exhibit low catalytic activity. In this study, a structure–function-guided computational approach was applied to identify a cytochrome P450 monooxygenase (CosaP450) with 11α-hydroxylation activity. By modulating the flexibility of the gating helix (N62-E75), the engineered variant MT3 (T72Q/F70T/Y226Q) increased the production of 11α,17α-hydroxyprogesterone (11α,17α-OHP) from 680 mg/L to 1140 mg/L. Then, the targeted engineering of the substrate channel and active site was employed based on the multiscale MD simulations, and the mutant MT6 (T72Q/F70T/Y226Q/V374A/G137T/E118G) achieved a yield of 1863 mg/L. MD analysis revealed that V374A relieved channel steric hindrance, while G137T/E118G remodeled the hydrogen-bond network to stabilize the binding conformation. Redox partner engineering further enhanced the titer of MT6 AnCPR to 2489 mg/L. This work establishes a computational framework for rational discovery and engineering of efficient 11α-hydroxylases, providing a promising strategy for enzymatic steroid functionalization.