Publications

• Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri.

  Alhapel A., Darley D.J., Wagener N., Eckel E., Elsner N., Pierik A.J.

  The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encod ... >> More

  The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria. << Less

  Proc. Natl. Acad. Sci. U.S.A. 103:12341-12346(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Nicotinic acid metabolism. 2,3-Dimethylmalate lyase.

  Pirzer P., Lill U., Eggerer H.

  1) A new enzyme, 2,3-dimethylmalate lyase, was purified from Clostridium barkeri to about 80% homogeneity. Some of the properties of the enzyme are described. 2) It is shown that the 2,3-dimethylmalic acid (m.p. 143 degrees C) described in the literature represents only one racemic pair. This pair ... >> More

  1) A new enzyme, 2,3-dimethylmalate lyase, was purified from Clostridium barkeri to about 80% homogeneity. Some of the properties of the enzyme are described. 2) It is shown that the 2,3-dimethylmalic acid (m.p. 143 degrees C) described in the literature represents only one racemic pair. This pair is not attacked by 2,3-dimethylmalate lyase. 3) The isolation of both racemic pairs of 2,3-dimethylmalic acid is described. Half of one pair, m.p. 104-106 degrees C, was converted to propionate and pyruvate by 2,3-dimethylmalate lyase. 4) In combination with earlier work performed by E.R. Stadtman and coworkers the results given under points 1--3 establish 2,3-dimethylmalate as an intermediate in the degradation of nicotinic acid by C. barkeri. 5) Experimental evidence indicates the 2,3-dimethylmalate lyase is no acyl-S-enzyme and that it is different in this respect as well as in quaternary structure from the apparently related enzymes citrate lyase and citramalate lyase. << Less

  Hoppe-Seyler's Z. Physiol. Chem. 360:1693-1702(1979) [PubMed] [EuropePMC]

• Nicotinic acid metabolism enzymic preparation and absolute configuration of the substrate for 2,3-dimethylmalate lyase.

  Lill U., Pirzer P., Kukla D., Huber R., Eggerer H.

  1) A convenient method for the enzymatic preparation of a chemically and optically pure isomer of 2,3-dimethylmalic acid in g-amounts is described. Propionate, pyruvate and partially purified 2,3-dimethylmalate lyase (from Clostridium barkeri) were applied. 2) The enzymically formed product, m.p. ... >> More

  1) A convenient method for the enzymatic preparation of a chemically and optically pure isomer of 2,3-dimethylmalic acid in g-amounts is described. Propionate, pyruvate and partially purified 2,3-dimethylmalate lyase (from Clostridium barkeri) were applied. 2) The enzymically formed product, m.p. 99--100 degrees C, [alpha]D20 = -16.4 (water), is related to the known stereochemistry of the Senecio alkaloid jacobine and to a laevorotatory 2,3-dimethylmalic acid derived from jaconecic acid, a degradation product of the alkaloid. From this relationship it appears likely that the substrate of the lyase is a component of the threo racemate and is of (2R,3S) configuration. 3) A three-dimensional X-ray structure analysis was performed and the structure refined to an R value of 0.049. The asymmetric unit contains three independent threo dimethylmalic acid molecules. The anomalous dispersion effects of carbon and oxygen were used to determine the absolute configuration. These measurements yielded a (2R,3S) configuration. 4) We conclude from these results that (2R,3S)-2,3-dimethylmalate is the substrate of the lyase. The results also establish that previously isolated racemic 2,3-dimethylmalic acids, m.p. 143 degrees C and m.p. 104--106 degrees C, represent the erythro and threo pair, respectively. << Less

  Hoppe-Seyler's Z. Physiol. Chem. 361:875-884(1980) [PubMed] [EuropePMC]