4 research outputs found

    Biosynthetic Versatility and Coordinated Action of 5′-Deoxyadenosyl Radicals in Deazaflavin Biosynthesis

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    Coenzyme F<sub>420</sub> is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F<sub>o</sub>) is still poorly understood. F<sub>o</sub> synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5′-deoxyadenosyl radicals to catalyze F<sub>o</sub> formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a <i>p</i>-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F<sub>o</sub> synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5′-deoxyadenosyl radicals to mediate the formation of a complex heterocycle

    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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