Discovery and Biosynthesis of Lanthipeptides Featuring an Azepinoindole Scaffold by Radical S-Adenosylmethionine Enzyme-Catalyzed C-C Bond Formation.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) continue to provide a rich source of structurally diverse and bioactive natural products, yet radical S-adenosylmethionine (rSAM)-catalyzed C-C bond formation remains unexplored in lanthipeptide biosynthesis. Here, we report the discovery of azepinopeptide A, a new class of lanthipeptides featuring an unprecedented tetrahydropyrrolo[1',2':1,2]azepino[3,4-b]indole scaffold formed through rSAM-catalyzed cross-linking of adjacent Trp1 and Pro2 residues. This unusual sp2-sp3 C-C bond formation, which has not been observed in any previously characterized rSAM enzyme-catalyzed RiPP, is mediated by a distinct subclass of lanthipeptide rSAM enzymes. Biochemical reconstitution and mutational analyses demonstrate that these enzymes exclusively recognize the mature, leader-free lanthipeptide substrate and lack both the RiPP recognition element and the auxiliary iron-sulfur cluster that are essential in other RiPP rSAM systems. Computational structural analysis, supported by in vivo co-expression studies, reveals a specialized binding pocket capable of accommodating the lanthipeptide substrate and positioning the catalytic [4Fe-4S] cluster deep within the pocket. This arrangement provides a basis for the observed regioselectivity and strict requirement for leader peptide removal. Azepinopeptides A and B exhibit potent neuroprotective activity at low concentrations comparable to that of the positive control, 3-n-butylphthalide. Bioinformatic analyses further uncover hundreds of related biosynthetic gene clusters, highlighting the prevalence of this biosynthetic strategy. Together, these findings expand the catalytic repertoire of rSAM enzymes and introduce a new platform for the biosynthetic generation of medium-sized ring peptide architectures with therapeutic potential.