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The biosynthesis of thiol- and thioether-containing cofactors and secondary metabolites catalyzed by radical S-adenosylmethionine enzymes.

ABSTRACT: Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links found in peptide natural products. Radical S-adenosyl-L-methionine (SAM) enzymes use an iron-sulfur cluster to catalyze the reduction of SAM to methionine and a highly reactive 5'-deoxyadenosyl radical; this radical can abstract hydrogen atoms at unreactive positions, facilitating the introduction of a variety of functional groups. Radical SAM enzymes that catalyze sulfur insertion reactions contain a second iron-sulfur cluster that facilitates the chemistry, either by donating the cluster's endogenous sulfide or by binding and activating exogenous sulfide or sulfur-containing substrates. The use of radical chemistry involving iron-sulfur clusters is an efficient anaerobic route to the generation of carbon-sulfur bonds in cofactors, secondary metabolites, and other natural products.

SUBMITTER: Jarrett JT

PROVIDER: S-EPMC4326807 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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The biosynthesis of thiol- and thioether-containing cofactors and secondary metabolites catalyzed by radical S-adenosylmethionine enzymes.

Jarrett Joseph T JT

The Journal of biological chemistry 20141204 7

Sulfur atoms are present as thiol and thioether functional groups in amino acids, coenzymes, cofactors, and various products of secondary metabolic pathways. The biosynthetic pathways for several sulfur-containing biomolecules require the substitution of sulfur for hydrogen at unreactive aliphatic or electron-rich aromatic carbon atoms. Examples discussed in this review include biotin, lipoic acid, methylthioether modifications found in some nucleic acids and proteins, and thioether cross-links ...[more]

PMID: 25477512

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Project description:The radical non-α-carbon thioether peptides (ranthipeptides) are a newly described class of ribosomally synthesized and post-translationally modified peptide (RiPP). Ranthipeptide biosynthetic gene clusters are characterized by a Cys-rich precursor peptide and a radical S-adenosylmethionine (rSAM)-dependent enzyme that forms a thioether linkage between a Cys donor and an acceptor residue. Unlike the sulfur-to-α-carbon linked thioether peptides (sactipeptides), known ranthipeptides contain thioethers to either the β- or γ-carbon (i.e., non-α-carbon) of an acceptor residue. Recently, we reported the discovery of freyrasin, a ranthipeptide from Paenibacillus polymyxa, which contains six thioethers from Cys-X3-Asp motifs present in the precursor peptide (PapA). The linkages are exclusively to the β-carbon of Asp (S-Cβ). In this report, we performed mutational analysis of PapA and the cognate thioether-forming rSAM enzyme (PapB) to define the substrate scope. Using a mass spectrometry-based activity assay, our data show that PapB is intolerant toward Ala and Asn in the acceptor position but tolerates Glu-containing variants. NMR spectroscopic data of a Glu variant demonstrated that the thioether linkage was to the 4-position of Glu (S-Cγ). Furthermore, we demonstrate that PapB is intolerant to expansion and contraction of the thioether motifs (Cys-Xn-Asp, n = 2 or 4), although a minimal substrate featuring only one Cys-X3-Asp motif was competent for thioether formation. Akin to the sactipeptides, PapB was dependent on a RiPP recognition element (RRE) to bind the cognate precursor peptide, with deletion resulting in loss-of-function in vivo. The activity of PapB could be restored in vivo by supplying the excised RRE in trans. Finally, we reconstituted the activity of PapB in vitro, which led to modification of all six Cys residues in PapA. These studies provide insights into ranthipeptide biosynthesis and expand our understanding of rSAM enzyme chemistry in natural product biosynthesis.

Project description:A [4Fe-4S](+) cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5'-deoxyadenosyl radical (5'-dA(•)). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5'-dA(•) is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, L-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5'-dA(•) to the product radical L-?-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the L-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN(-) ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp(2)-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.

Dataset Information

The biosynthesis of thiol- and thioether-containing cofactors and secondary metabolites catalyzed by radical S-adenosylmethionine enzymes.

Publications

The biosynthesis of thiol- and thioether-containing cofactors and secondary metabolites catalyzed by radical S-adenosylmethionine enzymes.

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