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Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

ABSTRACT: Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the β-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.

SUBMITTER: Eastman KAS

PROVIDER: S-EPMC10470005 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

Eastman Karsten A S KAS Jochimsen Andrew S AS Bandarian Vahe V

The Journal of biological chemistry 20230717 9

Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thio ...[more]

PMID: 37460016

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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-β-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.

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Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

Publications

Intermolecular electron transfer in radical SAM enzymes as a new paradigm for reductive activation.

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