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

Coenzyme F₄₂₀ 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_o) is still poorly understood. F_o synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5′-deoxyadenosyl radicals to catalyze F_o 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_o 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 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

Biosynthesis of F₀, Precursor of the F₄₂₀ Cofactor, Requires a Unique Two Radical-SAM Domain Enzyme and Tyrosine as Substrate

Cofactors play key roles in metabolic pathways. Among them F₄₂₀ 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, F₀-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 F₀-synthase and their <i>in vitro</i> activities. Our study allows us to establish that F₀-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 F₀-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

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

Radical <i>S</i>‑Adenosyl‑l‑Methionine Enzyme PylB: A C‑Centered Radical to Convert l‑Lysine into (3<i>R</i>)‑3-Methyl‑d‑Ornithine

PylB is a radical S-adenosyl-l-methionine (SAM) enzyme predicted to convert l-lysine into (3R)-3-methyl-d-ornithine, a precursor in the biosynthesis of the 22nd proteogenic amino acid pyrrolysine. This protein highly resembles that of the radical SAM tyrosine and tryptophan lyases, which activate their substrate by abstracting a H atom from the amino-nitrogen position. Here, combining in vitro assays, analytical methods, electron paramagnetic resonance spectroscopy, and theoretical methods, we demonstrated that instead, PylB activates its substrate by abstracting a H atom from the Cγ position of l-lysine to afford the radical-based β-scission. Strikingly, we also showed that PylB catalyzes the reverse reaction, converting (3R)-3-methyl-d-ornithine into l-lysine and using catalytic amounts of the 5′-deoxyadenosyl radical. Finally, we identified significant in vitro production of 5′-thioadenosine, an unexpected shunt product that we propose to result from the quenching of the 5′-deoxyadenosyl radical species by the nearby [Fe4S4] cluster