Discovery of biaryl macrocyclic peptides with C-terminal beta-keto acid groups

Advances in genome sequencing and bioinformatics methods have identified myriad biosynthetic gene clusters (BGCs) encoding uncharacterized molecules. By examining genomic databases for BGCs containing a prevalent peptide-binding domain used for the biosynthesis of most ribosomally synthesized and post-translationally modified peptides (RiPPs), we uncovered a new class involving modifications installed by a cytochrome P450, a multi-nuclear iron-dependent non-heme oxidase (MNIO, formerly DUF692), a cobalamin- and radical S-adenosyl-L-methionine-dependent enzyme (B12-rSAM), and a methyltransferase. After purification of the constituent enzymes, the biosynthetic pathway was reconstituted in vitro. Structural characterization demonstrated that a P450 catalyzed biaryl C-C crosslink formation between two Tyr residues. The B12-rSAM generated beta-methyltyrosine, while the MNIO transformed a C-terminal Asp residue into a C-terminal aminopyruvic acid. The methyltransferase acted on the beta-carbon of the α-keto acid. Marfey's method and exciton-coupled circular dichroism spectroscopy were used to elucidate the stereochemical configurations for the chiral carbons and the atropisomer that formed upon biaryl crosslinking. To the best of our knowledge, the MNIO featured in this biosynthetic pathway is the first to act on a non-Cys residue. Our study underscores that the pace of discovery of new macrocyclic peptides deriving from ribosomal peptides continues to accelerate and that RiPP BGCs remain a significant source of previously undiscovered enzyme chemistry.