Biosynthesis of macrocyclic peptides with C-terminal β-amino-α-keto acid groups by three different metalloenzymes

Abstract

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 approximately half of 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 oxidative enzyme (MNIO, formerly DUF692), a cobalamin- and radicalS-adenosyl-L-methioninedependent enzyme (B12-rSAM), and a methyltransferase. The activity of each enzyme was reconstitutedin vivousingBurkholderiasp. FERM BP-3421. Structural characterization demonstrated that the P450 catalyzed a biaryl C-C crosslink formation between two Tyr residues. The B12-rSAM generated β-methyltyrosine, while the MNIO transformed a C-terminal Asp residue into a C-terminal aminopyruvic acid. The methyltransferase acted on the β-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.

A widespread family of ribosomal peptide metallophores involved in bacterial adaptation to metal stress

“…recently to explore the chemical space of RiPPs involving MNIOs (24,25,28,30,47). To date, members of this superfamily of enzymes are known to catalyse remarkable oxidation reactions, including oxazolone coupled to thioamide formation, excision of cysteine β-methylene, Cysinvolved macrocyclization, and C-C and C-N cleavages, as evidenced in the biosynthesis of methanobactins, TglA-type pearlins, RiPPs in Chryseobacterium, aminopyruvatides, and methanobactin-like peptides in Vibrio, respectively (24,25,27,28,30). This work thus further expands the reaction spectrum of MNIOs.…”

“…Secondly, transformation of Cys to 5-thiooxazole is catalyzed by the MNIO enzyme BufB and its partner protein BufC, and not by a radical-SAM enzyme as was recently reported 33 . Intense efforts have been made recently to explore the chemical space of RiPPs involving MNIOs 15, 16, 19, 21, 44 . To date, members of this superfamily of enzymes are known to catalyze remarkable oxidation reactions, including oxazolone coupled to thioamide formation, excision of cysteine β-methylene, Cys-involved macrocyclization, and C-C and C-N cleavages, as evidenced in the biosynthesis of methanobactins, TglA-type pearlins, RiPPs in Chryseobacterium , aminopyruvatides, and methanobactin-like peptides in Vibrio , respectively 15, 16, 19–21 .…”

“…The other gig-like operons share the same gene organizations and predicted functions. In light of the recent discovery of MNIOs involved in RiPP biosynthesis (24)(25)(26)(27)(28)(29)(30), we hypothesized that BufB1 and BufC1 would install modifications on Cys residues of BufA1. Upstream of bufA1, CCNA_3362 and CCNA_3361 encoding the extracytoplasmic function (ECF)-family sigma factor SigF and the anti-sigma protein NrsF were also found to be induced by copper stress (19) and are therefore part of the buf1 BGC.…”

“…Parallel SSN analyses of the MNIO enzymes superfamily (Fig. 6C) showed that those associated with DF2282 or BUF_6/12Cys bufferins belong to the largest sequence cluster, distinct from smaller clusters of enzymes involved in the biosynthesis of methanobactins, RiPPs from Chryseobacterium, TglA-type pearlins and amino-pyruvatides (24,(26)(27)(28)30). Therefore, we have uncovered two large families of RiPPs whose biosynthesis involves MNIO enzymes, exemplified by the C. vibrioides bufferins, that are distinct from previously described MNIO enzyme-modified RiPPs.…”

“…The vast structural diversity of RiPPs is conferred by unlimited possibilities of chemical transformations. An emerging family of PTM enzymes is the DUF692 family of multinuclear non-heme iron dependent oxidases (MNIOs) that catalyse various reactions mostly but not exclusively on Cys residues, in the biosynthesis of specific RiPPs (24)(25)(26)(27)(28)(29)(30). However, the full spectrum of reactions catalysed by MNIO enzymes remains far from being fully appreciated.…”