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bioRxiv · 10.64898/2026.01.19.700444

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

Wang, B.·Knox, H. L.·York, N. J.·Radle, M. I.·Silakov, A.·Booker, S. J.
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Abstract

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 S-adenosylmethionine (SAM) methylase NosN. Here, we report three X-ray crystal structures of the NosN homolog, NocN, at resolutions of 1.4 [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 SAMI, which coordinates to the [Fe4S4] cluster, lies 3.5 [A] from the methyl group of SAMII 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 SAMII may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAMII-derived methylene radical to the MIA substrate.

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Wang, B., Knox, H. L., York, N. J., Radle, M. I., Silakov, A., Booker, S. J.. 2026-01-20. Structural and Spectroscopic Basis for Catalysis by a Class C Radical S-adenosylmethionine Methylase Involved in Nosiheptide/Nocathiacin Biosynthesis. https://doi.org/10.64898/2026.01.19.700444

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