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Mechanistic elucidation of the mycofactocin-biosynthetic radical S-adenosylmethionine protein, MftC.

ABSTRACT: Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depends on radical S-adenosylmethionine proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an S-adenosylmethionine protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr-30) on the mycofactocin precursor peptide MftA; however, this product has not been verified by techniques other than MS. Herein, we provide a more detailed study of MftC catalysis and report a revised mechanism for MftC chemistry. We show that MftC catalyzes the formation of two isomeric products. Using a combination of MS, isotope labeling, and 1H and 13C NMR techniques, we established that the major product, MftA*, is a tyramine-valine-cross-linked peptide formed by MftC through two S-adenosylmethionine-dependent turnovers. In addition, we show that the hydroxyl group on MftA Tyr-30 is required for MftC catalysis. Furthermore, we show that a substitution in the penultimate MftA Val-29 position causes the accumulation of an MftA** minor product. The 1H NMR spectrum indicates that this minor product contains an ??-unsaturated bond that likely arises from an aborted intermediate of MftA* synthesis. The finding that MftA* is the major product formed during MftC catalysis could have implications for the further elucidation of mycofactocin biosynthesis.

SUBMITTER: Khaliullin B

PROVIDER: S-EPMC5546040 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Mechanistic elucidation of the mycofactocin-biosynthetic radical S-adenosylmethionine protein, MftC.

Khaliullin Bulat B Ayikpoe Richard R Tuttle Mason M Latham John A JA

The Journal of biological chemistry 20170620 31

Ribosomally synthesized and posttranslationally modified peptide (RiPP) pathways produce a diverse array of natural products. A subset of these pathways depends on radical S-adenosylmethionine proteins to modify the RiPP-produced peptide. Mycofactocin biosynthesis is one example of an S-adenosylmethionine protein-dependent RiPP pathway. Recently, it has been shown that MftC catalyzes the oxidative decarboxylation of the C-terminal tyrosine (Tyr-30) on the mycofactocin precursor pep ...[more]

PMID: 28634235

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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

Mechanistic elucidation of the mycofactocin-biosynthetic radical S-adenosylmethionine protein, MftC.

Publications

Mechanistic elucidation of the mycofactocin-biosynthetic radical <i>S</i>-adenosylmethionine protein, MftC.

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