Hydroxylation and Biological Activity Evaluation of Isosteviol C12 by P450 BM3

Isosteviol, a naturally occurring tetracyclic diterpenoid, is a multifunctional compound exhibiting a wide range of biological activities. The activation of inert C─H bonds in isosteviol holds significant potential for enhancing its biological efficacy; however, this process remains a formidable challenge in conventional organic synthesis. Cytochrome P450 BM3, a well-established biocatalyst, is frequently employed to address this challenge. In this study, we utilized compound similarity analysis, molecular docking, and site-directed mutagenesis to successfully achieve the regioselective hydroxylation of isosteviol at the 12β position, yielding 12β-hydroxyisosteviol, catalyzed by P450 BM3. Comparative analyses revealed that 12β-hydroxyisosteviol exhibits superior anticancer and anti-inflammatory properties relative to its precursor, isosteviol. Through systematic site-directed mutagenesis targeting eight potential key amino acid residues in the BM3 variant, we constructed a focused mutant library. Among these, the engineered mutant M05 (F87A/A330W/A82G) demonstrated enhanced catalytic efficiency, increasing the conversion rate of isosteviol from 59% to 66%. This study highlights the potential of enzyme engineering for the selective functionalization of complex natural products.