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Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis.

ABSTRACT: The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the beta-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylthienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, thienamycin and N-acetylthienamycin, in a process likely to be general for the broader class of these antibiotics.

SUBMITTER: Freeman MF

PROVIDER: S-EPMC2516261 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis.

Freeman Michael F MF Moshos Kristos A KA Bodner Micah J MJ Li Rongfeng R Townsend Craig A CA

Proceedings of the National Academy of Sciences of the United States of America 20080804 32

The enzymatic activities of three proteins encoded by the thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the beta-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopante ...[more]

PMID: 18678912

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Project description:Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420 Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future.IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Dataset Information

Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis.

Publications

Four enzymes define the incorporation of coenzyme A in thienamycin biosynthesis.

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