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

A Radical Transfer Pathway in Spore Photoproduct Lyase

Spore photoproduct lyase (SPL) repairs a covalent UV-induced thymine dimer, spore photoproduct (SP), in germinating endospores and is responsible for the strong UV resistance of endospores. SPL is a radical <i>S</i>-adenosyl-l-methionine (SAM) enzyme, which uses a [4Fe-4S]⁺ cluster to reduce SAM, generating a catalytic 5′-deoxyadenosyl radical (5′-dA^•). This in turn abstracts a H atom from SP, generating an SP radical that undergoes β scission to form a repaired 5′-thymine and a 3′-thymine allylic radical. Recent biochemical and structural data suggest that a conserved cysteine donates a H atom to the thymine radical, resulting in a putative thiyl radical. Here we present structural and biochemical data that suggest that two conserved tyrosines are also critical in enzyme catalysis. One [Y99_(<i>Bs</i>) in <i>Bacillus subtilis</i> SPL] is downstream of the cysteine, suggesting that SPL uses a novel hydrogen atom transfer (HAT) pathway with a pair of cysteine and tyrosine residues to regenerate SAM. The other tyrosine [Y97_(<i>Bs</i>)] has a structural role to facilitate SAM binding; it may also contribute to the SAM regeneration process by interacting with the putative ^•Y99_(<i>Bs</i>) and/or 5′-dA^• intermediates to lower the energy barrier for the second H abstraction step. Our results indicate that SPL is the first member of the radical SAM superfamily (comprising more than 44000 members) to bear a catalytically operating HAT chain

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