The B₁₂-Radical SAM Enzyme PoyC Catalyzes Valine C_β‑Methylation during Polytheonamide Biosynthesis

Genomic and metagenomic investigations have recently led to the delineation of a novel class of natural products called ribosomally synthesized and post-translationally modified peptides (RiPPs). RiPPs are ubiquitous among living organisms and include pharmaceutically relevant compounds such as antibiotics and toxins. A prominent example is polytheonamide A, which exhibits numerous post-translational modifications, some of which were unknown in ribosomal peptides until recently. Among these post-translational modifications, C-methylations have been proposed to be catalyzed by two putative radical <i>S</i>-adenosylmethionine (rSAM) enzymes, PoyB and PoyC. Here we report the <i>in vitro</i> activity of PoyC, the first B₁₂-dependent rSAM enzyme catalyzing peptide C_β-methylation. We show that PoyC catalyzes the formation of <i>S</i>-adenosylhomocysteine and 5′-deoxyadenosine and the transfer of a methyl group to l-valine residue. In addition, we demonstrate for the first time that B₁₂-rSAM enzymes have a tightly bound MeCbl cofactor that during catalysis transfers a methyl group originating from <i>S</i>-adenosyl-l-methionine. Collectively, our results shed new light on polytheonamide biosynthesis and the large and emerging family of B₁₂-rSAM enzymes

Mechanistic Investigations of PoyD, a Radical <i>S</i>‑Adenosyl‑l‑methionine Enzyme Catalyzing Iterative and Directional Epimerizations in Polytheonamide A Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of bioactive peptides. Among RiPPs, the bacterial toxin polytheonamide A is characterized by a unique set of post-translational modifications catalyzed by novel radical <i>S</i>-adenosyl-l-methionine (SAM) enzymes. Here we show that the radical SAM enzyme PoyD catalyzes in vitro polytheonamide epimerization in a <i>C</i>-to-<i>N</i> directional manner. By combining mutagenesis experiments with labeling studies and investigating the enzyme substrate promiscuity, we deciphered in detail the mechanism of PoyD. We notably identified a critical cysteine residue as a likely key H atom donor and demonstrated that PoyD belongs to a distinct family of radical SAM peptidyl epimerases. In addition, our study shows that the core peptide directly influences the epimerization pattern allowing for production of peptides with unnatural epimerization patterns

Rational Design of a Low Molecular Weight, Stable, Potent, and Long-Lasting GPR103 Aza‑β³‑pseudopeptide Agonist

26RFa, a novel RFamide neuropeptide, is the endogenous ligand of the former orphan receptor GPR103. Intracerebroventricular injection of 26RFa and its C-terminal heptapeptide, 26RFa_(20–26), stimulates food intake in rodents. To develop potent, stable ligands of GPR103 with low molecular weight, we have designed a series of aza-β³-containing 26RFa_(20–26) analogues for their propensity to establish intramolecular hydrogen bonds, and we have evaluated their ability to increase [Ca²⁺]_i in GPR103-transfected cells. We have identified a compound, [Cmpi²¹,aza-β³-Hht²³]­26RFa_(21–26), which was 8-fold more potent than 26RFa_(20–26) in mobilizing [Ca²⁺]_i. This pseudopeptide was more stable in serum than 26RFa_(20–26) and exerted a longer lasting orexigenic effect in mice. This study constitutes an important step toward the development of 26RFa analogues that could prove useful for the treatment of feeding disorders