Radical S-Adenosyl-l-Methionine Oxygenase DarE Forms Ether Bond via a Partially Delocalized Tryptophan C_β Radical

Darobactins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against Gram-negative pathogens. Their antibiotic function depends on a distinctive β-strand architecture stabilized by an ether cross-link between two tryptophan residues. Unlike most ether linkages in natural products, which arise from hydroxylated precursors, the darobactin ether bond is formed between two unmodified Trp residues by the radical S-adenosyl-l-methionine (SAM) enzyme DarE. DarE catalyzes both O-insertion and ether bond formation using molecular oxygen as a cosubstrate, but its catalytic mechanism remains largely unexplored. Here, we report detection and characterization of a key radical intermediate at the C_β position of W3 (W3-C_β•) using isotopically labeled DarA, kinetic analysis, and spectroscopy methods including EPR, ENDOR, and HYSCORE. Similar radical species were also observed in reactions with W3 variants of DarA. These results provide direct evidence that DarE catalyzes ether cross-link formation through reaction of the W3-C_β• intermediate with O₂, revealing an unprecedented radical-mediated strategy for O-atom incorporation in RiPP biosynthesis.