Discovery and Biosynthesis of Cihanmycins Reveal Cytochrome P450-Catalyzed Intramolecular C-O Phenol Coupling Reactions.

Cinnamoyl-containing nonribosomal peptides (CCNPs) constitute a unique family of natural products. The enzyme mechanism for the biaryl phenol coupling reaction of the bicyclic CCNPs remains unclear. Herein, we report the discovery of two new arabinofuranosylated bicyclic CCNPs cihanmycins (CHMs) A () and B () from DSM 45679 and the identification of the CHM biosynthetic gene cluster ( BGC) by heterologous expression in SBT18 to afford CHMs C () and D (). The structure of was confirmed by X-ray diffraction analysis. Three cytochrome P450 enzyme (CYP)-encoding genes , , and were individually inactivated in the heterologous host to produce CHMs E (), F (), and G (), respectively. The structures of and indicated that Cih26 was responsible for the hydroxylation and epoxidation of the cinnamoyl moiety, and Cih32 should catalyze the β-hydroxylation of three amino acid residues. Cih33 and its homologues DmlH and EpcH were biochemically verified to convert CHM G () with a monocyclic structure to a bicyclic skeleton of CHM C () through an intramolecular C-O phenol coupling reaction. The substrate -bound crystal structure of DmlH not only established the structure of , which was difficult for NMR analysis for displaying anomalous splitting signals, but also provided the binding mode of macrocyclic peptides recognized by these intramolecular C-O coupling CYPs. In addition, computational studies revealed a water-mediated diradical mechanism for the C-O phenol coupling reaction. These findings have shed important mechanistic insights into the CYP-catalyzed phenol coupling reactions.