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Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

ABSTRACT: We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel β-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA. The latter γ-diketo CoA ester undergoes two parallel cyclization processes. One process yields a mixture of tautomers, i.e., cyclopentenyl- and cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the ε-nitrogen of lysine but without carbons from lysine. The cyclic CoA esters were identified in rat livers perfused with levulinate and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Lastly, 3,6-diketoheptanoyl-CoA, like 2,5-diketohexane, pyrrolates free lysine and, presumably, lysine residues from proteins. This may represent a new pathway for protein pyrrolation. The cyclic CoA esters and related pyrrolation processes may play a role in the toxic effects of 4-hydroxypentanoate.

SUBMITTER: Harris SR

PROVIDER: S-EPMC3997259 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

Harris Stephanie R SR Zhang Guo-Fang GF Sadhukhan Sushabhan S Wang Hua H Shi Chuan C Puchowicz Michelle A MA Anderson Vernon E VE Salomon Robert G RG Tochtrop Gregory P GP Brunengraber Henri H

Chemical research in toxicology 20121206 2

We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel β-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to ...[more]

PMID: 23171137

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Project description:Clorobiocin is a well-known, highly effective inhibitor of DNA gyrase belonging to the aminocoumarin antibiotics. To identify potentially novel derivatives of this natural product, we conducted an untargeted investigation of clorobiocin biosynthesis in the known producer Streptomyces roseochromogenes DS 12.976 using LC-MSE, molecular networking, and analysis of fragmentation spectra. Previously undescribed clorobiocin derivatives uncovered in this study include bromobiocin, a variant halogenated with bromine instead of chlorine, hydroxylated clorobiocin, carrying an additional hydroxyl group on its 5-methyl-pyrrole 2-carboxyl moiety, and two other derivatives with modifications on their 3-dimethylallyl 4-hydroxybenzoate moieties. Furthermore, we identified several compounds not previously considered clorobiocin pathway products, which provide new insights into the clorobiocin biosynthetic pathway. By supplementing the medium with different concentrations of potassium bromide, we confirmed that the clorobiocin halogenase can utilize bromine instead of chlorine. The reaction, however, is impeded such that non-halogenated clorobiocin derivatives accumulate. Preliminary assays indicate that the antibacterial activity of bromobioin against Bacillus subtilis and efflux-impaired Escherichia coli matches that of clorobiocin. Our findings emphasize that yet unexplored compounds can be discovered from established strains and biosynthetic gene clusters by means of metabolomics analysis and highlight the utility of LC-MSE-based methods to contribute to unraveling natural product biosynthetic pathways.ImportanceThe aminocoumarin clorobiocin is a well-known gyrase inhibitor produced by the gram-positive bacterium Streptomyces roseochromogenes DS 12.976. To gain a deeper understanding of the biosynthetic pathway of this complex composite of three chemically distinct entities and the product spectrum, we chose a metabolite-centric approach. Employing high-resolution LC-MSE analysis, we investigated the pathway products in extracted culture supernatants of the natural producer. Novel pathway products were identified that expand our understanding of three aspects of the biosynthetic pathway, namely the modification of the noviose, transfer and methylation of the pyrrole 2-carboxyl moiety, and halogenation. For the first time, brominated products were detected. Their levels and the levels of non-halogenated products increased in medium supplemented with KBr. Based on the presented data, we propose that the enzyme promiscuity contributes to a broad product spectrum.

Dataset Information

Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

Publications

Metabolomics and mass isotopomer analysis as a strategy for pathway discovery: pyrrolyl and cyclopentenyl derivatives of the pro-drug of abuse, levulinate.

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