An Enzyme from a Single P450 Monooxygenase Clade Orchestrates Six‑Electron Oxidation for Cyclopeptide Modification

Abstract

The oxidative modification of complex natural products is critical for their biological activity. However, the enzymatic mechanisms that facilitate three-step, six-electron transfer reactions, such as the conversion of a methyl group to a carboxylic acid, on cyclopeptide scaffolds remain poorly understood. This study characterizes IlaL, a cytochrome P450 monooxygenase from Streptomyces atratus, which catalyzes a six‑electron methyl‑to‑carboxylic acid conversion during the biosynthesis of the potent antimycobacterial cyclopeptide ilamycins. IlaL possesses a noncanonical “Asp-Asn (DN)” dioxygen activation dyad and an extended, flexible BC-loop, which together enable sequential three-step oxidation. The high-resolution co-crystal structure of IlaL bound to the substrate ilamycin B2 reveals an enlarged cavity and dynamic gating conferring precise regioselectivity and broad substrate tolerance. Biochemical and phylogenetic analyses confirm the essential role of the DN dyad and show that DN‑type cytochrome P450 monooxygenases occupy a distinct, recently emerged phylogenetic clade. These findings provide structural and mechanistic insights into six‑electron oxidative modification and establish design principles for versatile biocatalysts enabling late‑stage natural product diversification.