Structural and Spectroscopic Basis for Catalysis by a Class C Radical <i>S</i> -adenosylmethionine Methylase Involved in Nosiheptide/Nocathiacin Biosynthesis

Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits potent antibiotic activity against multiple bacterial pathogens. NOS features a core macrocyclic peptide containing thiazoles, dehydrated serine and threonine residues, and a 3-hydroxypyridine ring. In addition to the macrocycle, NOS possesses a side-ring system formed by a 3-methyl-2-indolic acid (MIA) bridge that connects to glutamyl and cysteinyl residues on the core peptide via ester and thioester linkages. This unique side-ring is installed by the class C radical <i>S</i>-adenosylmethionine (SAM) methylase NosN. Here, we report X-ray crystal structures of the NosN homolog NocN-the first structure of a class C radical SAM methylase. The structures reveal clear electron density for two bound SAM molecules. Remarkably, the C5' atom of SAM^I, which coordinates to the [Fe₄S₄] cluster, lies 3.1 Å from the methyl group of SAM^II and is properly positioned for direct hydrogen atom abstraction. A structure containing a product mimic illustrates how NocN engages its substrate and identifies Tyr276 as a key catalytic residue. The structure further suggests that the sulfonium center of SAM^II may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAM^II-derived methylene radical to the MIA substrate.