Cytochrome P450 Mediated Cyclohexane Ring Formation in Forazoline Biosynthesis

Abstract Forazoline A, produced by the marine actinomycete Actinomadura sp. WMMB‐499, is a unique PK/NRP hybrid macrolactone with promising antifungal in vivo efficacy through a previously unreported mechanism. Although a PKS/NRPS gene cluster was identified as a candidate for forazoline production, the precise biosynthetic pathway and the functions of the tailoring enzymes remain unclear. In this work, the functions of three cytochrome P450 mono‐oxygenases (FrazP1P2P3) were characterized. Notably, FrazP2 was found to mediate cyclohexane ring formation from an 1,3,6‐triene precursor during forazoline A biosynthesis, as confirmed by genetic and biochemical analysis. To gain structural and mechanistic insight into the activity of FrazP2, the crystal structure of a FrazP2‐substrate complex has been solved at 2.3 Å resolution. The molecular dynamics simulations and DFT calculations revealed an unprecedented enzyme‐catalyzed oxidative cyclization reaction by FrazP2. These findings expand our understanding of the catalytic diversity of cytochrome P450s, contributing to the diversification of natural products and enabling the creation of unnatural derivatives with increased antifungal potency.