4 research outputs found

    The B<sub>12</sub>-Radical SAM Enzyme PoyC Catalyzes Valine C<sub>β</sub>‑Methylation during Polytheonamide Biosynthesis

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    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 B<sub>12</sub>-dependent rSAM enzyme catalyzing peptide C<sub>β</sub>-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 B<sub>12</sub>-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 B<sub>12</sub>-rSAM enzymes

    Biosynthesis of F<sub>0</sub>, Precursor of the F<sub>420</sub> Cofactor, Requires a Unique Two Radical-SAM Domain Enzyme and Tyrosine as Substrate

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    Cofactors play key roles in metabolic pathways. Among them F<sub>420</sub> has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F<sub>0</sub>-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F<sub>0</sub>-synthase and their <i>in vitro</i> activities. Our study allows us to establish that F<sub>0</sub>-synthase, from both types, uses 5-amino-6-ribitylamino-2,4­(1<i>H</i>,3<i>H</i>)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F<sub>0</sub>-synthase generates two 5′-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes

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

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    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
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