Rationally engineering an H2O2-dependent P450 dihydroxylase for steroid functionalisation†

P450-catalysed steroid hydroxylation serves as both a fundamental biochemical pathway for in vivo steroid hormone biosynthesis and metabolism, and a pivotal tool for the biotechnological production of steroidal pharmaceuticals. Herein, we report the construction of an efficient H₂O₂-dependent P450 steroid dihydroxylase through rational engineering of the H₂O₂ tunnel, guided by molecular dynamics (MD) simulations and crystallographic analysis. The triple mutant F184A/F191A/E196A demonstrated an approximately 80-fold enhancement in catalytic efficiency (k_cat/K_m) for testosterone hydroxylation compared to wild-type CYP105D18, indicating a dramatic improvement in peroxygenase activity. Testosterone hydroxylation by this mutant predominantly yielded 2β-hydroxytestosterone (81%), with minor 16α-hydroxytestosterone (19%). Notably, the 2β-hydroxylated product could be quantitatively converted to 2β,15α-dihydroxytestosterone in the subsequent reaction. This study provides novel insights into the stepwise design of H₂O/H₂O₂ tunnels in P450 enzymes through the integration of MD simulations and crystallographic data. Furthermore, it establishes a practical enzymatic approach for the regio- and stereoselective dihydroxylation of steroids, with potential applications in pharmaceutical synthesis.