Biosynthesis of Macrocyclic Peptides with C‑Terminal β‑Amino-α-keto Acid Groups by Three Different Metalloenzymes

Advances in genome sequencing and bioinformatics methods
have identified
a myriad of biosynthetic gene clusters (BGCs) encoding uncharacterized
molecules. By mining genomes for BGCs containing a prevalent peptide-binding
domain used for the biosynthesis of ribosomally synthesized and post-translationally
modified peptides (RiPPs), we uncovered a new compound class involving
modifications installed by a cytochrome P450, a multinuclear iron-dependent
non-heme oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and
radical S-adenosyl-l-methionine-dependent
enzyme (B12-rSAM), and a methyltransferase. All enzymes were functionally
expressed in Burkholderia sp. FERM BP-3421. Structural
characterization demonstrated that the P450 enzyme catalyzed the formation
of a biaryl C–C cross-link between two Tyr residues with the
B12-rSAM generating β-methyltyrosine. The MNIO transformed a
C-terminal Asp residue into aminopyruvic acid, while the methyltransferase
acted on the β-carbon of this α-keto acid. Exciton-coupled
circular dichroism spectroscopy and microcrystal electron diffraction
(MicroED) were used to elucidate the stereochemical configuration
of the atropisomer formed upon biaryl cross-linking. To the best of
our knowledge, the MNIO featured in this pathway is the first to modify
a residue other than Cys. This study underscores the utility of genome
mining to isolate new macrocyclic RiPPs biosynthesized via previously
undiscovered enzyme chemistry.