Radical SAM Enzymes Catalyze Aromatic-Proline Cross-Links in Lanthipeptide Biosynthesis

Radical S-adenosylmethionine (rSAM) enzymes enable some of the most chemically challenging transformations in natural product biosynthesis, profoundly shaping the structural diversity of ribosomally synthesized and post-translationally modified peptides (RiPPs). Despite extensive studies in several RiPP families, the contribution of rSAM chemistry to lanthipeptide biosynthesis has remained poorly defined. Here, we identify a widespread group of rSAM enzymes associated with type III/IV lanthipeptide gene clusters through combined genome mining and phylogenetic analysis. Characterization of representative pathways uncovered a series of new lanthipeptides bearing an N-terminal aromatic-proline carbon-carbon cross-link. Detailed LC-MS/MS and NMR analyses revealed that Trp, Phe, or His residues are covalently linked to a conserved Pro residue, establishing a broader family of rSAM-catalyzed aromatic-proline couplings in lanthipeptide biosynthesis. Notably, biochemical reconstitution, active-site mutagenesis, substrate profiling, and isotope-labeling experiments define key catalytic requirements, support a radical-mediated dehydrogenative coupling mechanism, and show that lanthionine formation is not an absolute prerequisite for AitR-catalyzed aromatic-proline cross-linking. These findings broaden the substrate and structural scope of rSAM-catalyzed cross-linking in lanthipeptide RiPPs.