Radical-Mediated Nucleophilic Peptide Cross-Linking in Dynobactin Biosynthesis

Dynobactins are recently discovered ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics that selectively kill Gram-negative pathogens by inhibiting the β-barrel assembly machinery (Bam) located on their outer membranes. Such activity of dynobactins derives from their unique cross-links between Trp1-Asn4 and His6-Tyr8. In particular, the His6-Tyr8 cross-link is formed between Nτ of His6 and Cβ of Tyr8, an unprecedented type of cross-link in RiPP natural products. The mechanism of the C–N cross-link formation remains elusive. In this work, using in vitro characterizations, we demonstrate that both cross-links in dynobactins are biosynthesized by the radical S-adenosylmethionine (SAM) enzyme DynA. Subsequent mechanistic studies using deuterium-labeled DynB precursor peptides suggested that the C–N cross-linking proceeds through the Tyr8-Hβ atom abstraction by 5′-deoxyadenosyl radical. The absence of solvent exchange of Tyr8-Hα suggested that the mechanism unlikely involves α,β-desaturation of Tyr8. Furthermore, DynA catalyzed covalent modification of Tyr8 of H6A-DynB with small-molecule nucleophiles, suggesting the presence of a highly electrophilic Tyr-derived intermediate. Based on all these observations, we propose that DynA catalyzes Tyr8-Hβ atom abstraction to generate Tyr8-Cβ radical followed by its oxidation to a p-quinone methide intermediate, to which His6-Nτ attacks to form the C–N cross-link. This quinone methide-dependent mechanism of RiPPs cross-linking is distinct from the previously reported RiPPs cross-linking mechanisms and represents a novel mechanism in RiPPs biosynthesis. We will also discuss the functional, mechanistic, and evolutional relationships of DynA with other peptide-modifying radical SAM enzymes.