Improved 2α-Hydroxylation Efficiency of Steroids by CYP154C2 Using Structure-Guided Rational Design

This study focused on improving the 2α-hydroxylation efficiency of the enzyme CYP154C2 from Streptomyces avermitilis MA-4680T for the steroids androstenedione (ASD) and testosterone (TES). By solving the TES-bound structure of CYP154C2 at 1.42 Å, researchers designed eight mutants to enhance conversion efficiency. Notably, the L88F/M191F and M191F/V285L mutants significantly increased conversion rates for TES (8.9-fold and 7.4-fold) and ASD (46.5-fold and 19.5-fold) compared to the wild-type enzyme, while maintaining high selectivity for the 2α-position. The L88F/M191F mutant also showed improved substrate binding affinity. The study highlighted the role of L88 in substrate selectivity, as mutants with L88F produced 16α-hydroxylation products. This research provided a strategy for enhancing steroid hydroxylation efficiency, which is crucial for steroid drug biosynthesis.