Structure-Guided Engineering of CYP109B4: From Monooxygenase to Peroxygenase for Selective Steroid Hydroxylation

Cytochrome P450 CYP109B4 is a characterized 16β-steroid hydroxylase with significant potential in steroid drug biosynthesis, but its application is limited by reliance on costly coenzymes and complex electron transfer processes. To address this, we constructed a hydrogen peroxide (H 2 O 2 )-driven catalytic system through structure-guided engineering. Screening a combinatorial variant library identified the L240V/S387F double mutant, which exhibited emergent peroxygenase activity (baseline TTN = 11). Using focused rational iterative site-specific mutagenesis (FRISM), we obtained optimized variant B4–Pm9 exhibiting a 4-fold higher total turnover number (TTN = 44). Coupling with an alcohol dehydrogenase (Aldo)-based H 2 O 2 regeneration system further enhanced catalytic efficiency, achieving a TTN of 348 (31-fold improvement over L240V/S387F mutant). Computational studies revealed the H 2 O 2 activation mechanism in Pm7 and Pm9. Notably, variant B4–Pm7 demonstrated a novel mechanism of H 2 O 2 cleavage mediated by the “alcohol-hydroxyl” moiety, showcasing a significant breakthrough in the realm of P450 peroxygenase.