Dataset Information

Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S-Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System.

ABSTRACT: Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosM gene, the biosynthesis of the side-ring structure requires the actions of NosI, -J, -K, -L, and -N. NosN is annotated as a class C radical S-adenosylmethionine (SAM) methylase, but its true function is to transfer a C1 unit from SAM to C4 of 3-methyl-2-indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. However, exactly when NosN performs its function during the biosynthesis of nosiheptide is unknown. Herein, we report the syntheses and use of three peptide mimics as potential substrates designed to address the timing of NosN's function. Our results show that NosN clearly closes the side ring before NosO forms the pyridine ring and most likely before NosD/E catalyzes formation of the dehydrated amino acids, although the possibility of a more random process (i.e., NosN acting after NosD/E) cannot be ruled out. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction-based adducts containing SAM fused to C4 of MIA. The two SAM adducts are derived from a consensus radical-containing species proposed to be the key intermediate-or a derivative of the key intermediate-in our proposed catalytic mechanism of NosN.

SUBMITTER: Wang B

PROVIDER: S-EPMC7061316 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Publications

Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S-Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System.

Wang Bo B LaMattina Joseph W JW Marshall Savannah L SL Booker Squire J SJ

Journal of the American Chemical Society 20190401 14

Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosM gene, the ...[more]

PMID: 30865439

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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.

Dataset Information

Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S-Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System.

Publications

Capturing Intermediates in the Reaction Catalyzed by NosN, a Class C Radical S-Adenosylmethionine Methylase Involved in the Biosynthesis of the Nosiheptide Side-Ring System.

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