Abstact

Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide 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 S-adenosylmethionine (SAM) methylase NosN. Here, we report three X-ray crystal structures of the NosN homologue, NocN, at resolutions of 1.40 A, 1.84 A, and 1.78 A under anaerobic conditions, representing the first structural characterization 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 [Fe(4)S(4)] cluster, lies 3.5 A 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.