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 sp 2 –sp 3 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.