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PubMed1981

The catalytic mechanism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. One-electron transfer at two distinct steps of the catalytic cycle.

The catalytic cycle of the 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium was investigated. The first step is binding of the 2-oxoacid to the enzyme followed by decarboxylation and transfer of one electron to the [4Fe-4S] cluster of the functional unit. The cluster is then reoxidized by ferredoxin or, in the absence of the physiological electron acceptor, by oxygen. In the resulting stable enzyme-intermediate radical the decarboxylation product of the 2-oxoacid remains tightly bound until reaction with coenzyme A caused formation of acyl-CoA and concomitant transfer of the second electron to the cluster, which again is reoxidized by ferredoxin or oxygen. After purification, part of the enzyme molecules still contain the intermediate radical. Enzyme preparations either free of radical or containing enhanced amounts are obtained by treatment with coenzyme A or 2-oxoacid, respectively. Whenever the radical is present in an enzyme molecule the respective binding site for the 2-oxoacid is blocked.

Binding Sites↗
PubMed2001

Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.

In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.

Acetyl Coenzyme A↗
PubMed1982

Pyruvate oxidation by the Reiter strain of Treponema phagedenis.

Spectrophotometric assays of pyruvate oxidation catalyzed by extracts of the Reiter strain of Treponema phagedenis indicated that viologen dyes, flavin nucleotides, and a ferric iron chelate, but not pyridine nucleotides, were utilized as electron acceptors. Benzyl viologen-linked activity partially sedimented during ultracentrifugation and appeared similar to clostridial pyruvate:ferredoxin oxidoreductase with respect to the spectral properties of the enzyme chromophore. Electron carrier activity in treponemal extracts was quantitated by a metronidazole-linked assay in which the oxidation of pyruvate by carrier-depleted extracts led to the reduction of electron carrier in the crude extracts which then reduced metronidazole. The rate of metronidazole reduction was proportional to the amount of electron carrier present in the assay. Electron carrier activity in Triton X-100-solubilized, crude extracts partially purified by DEAE-cellulose chromatography and gel filtration was attributed to a protein possessing the spectral and physical properties of a ferredoxin. A similar protein appeared to be present in extracts of Treponema denticola ST10.

Electron Transport↗
PubMed2003

Attempts to inhibit ruminal methanogenesis by blocking pyruvate oxidative decarboxylation.

The inhibition of pyruvate oxidative decarboxylation as a means of decreasing ruminal methanogenesis in vitro was studied. In the first experiment, the addition of adenosine and adenine (with and without ribose) to ruminal batch cultures did not decrease methanogenesis. In the second experiment, the addition of oxythiamin decreased methanogenesis by 23%. In the third experiment, three pyruvate derivatives did not inhibit methanogenesis, although hydroxypyruvate improved organic matter fermentation from 57.8% to 64.2%. The additives did not seem to inhibit pyruvate oxidative decarboxylation.

Animals↗
PubMed1988

Muscle composition and its relation to sprint running in the lizard Dipsosaurus dorsalis.

Iguanid lizards exhibit considerable intraspecific variation in several aspects of their muscle composition. To determine the relationship of this variation to the variation in locomotor performance, running speeds of 20 male desert iguanas (Dipsosaurus dorsalis) of similar mass were measured from video recordings of animals as they sprinted down a 4.9-m runway maintained at 40 degrees C, the preferred body temperature of Dipsosaurus. Mean sprint speed ranged from 2.2 to 4.2 m/s. Selected muscles from these animals were then analyzed histochemically for fiber type size and composition, and the activities of citrate synthase, pyruvate kinase, and creatine kinase were measured. Muscle fiber cross-sectional areas were highly correlated within individuals, in three leg muscles and across all three fiber types, so that individuals could be characterized as possessing large or small fibers relative to the sample mean. Activities of all three enzymes also covaried within individuals so that individual lizards could be characterized as possessing high or low leg muscle catabolic capacity. There existed a significant and inverse relationship between fiber cross-sectional areas and muscle enzyme activities so that individuals with small muscle fibers tended to have higher catabolic capacities. Approximately 25-30% of the variation in mean sprint running speed could be predicted by variation in muscle fiber areas alone. The use of muscle fiber areas and snout vent length as independent variables in a multiple-regression equation explained approximately 50% of the sprint-running variation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

[Acetate photometabolism in Ectothiorhodospira shaposhnikovii].

The cells of Ectothiorhodospira were cultivated under autotrophic conditions and assimilated 14C-acetate in the presence of bicarbonate. The label was incorporated rapidly into phosphoglyceric acid (PGA) and phosphorous esters (PE) of sugars, into compounds of the tricarboxylic acid cycle (TAC), aspartate, glutamate. After some time the content of the label became the highest in glutamate and decreased in other compounds. The same products were formed upon the assimilation of acetate by the cells cultivated in its presence. However, the amount of PGA and PE of sugars, especially those formed in the presence of sulphides, was less, and their curve had a positive slope. Fluoroacetate inhibited the incorporation of 14C from acetate in the cells and caused an increase of labeled citrate. Iodacetate inhibited almost completely the fixation of CO2 by the cells in the presence of sulphide; the fixation of carbon dioxide constituted about 60 percent of the control if both sulphide and acetate were present in the medium. Therefore, the assimilation of acetate by Ect. shaposhnikovii can be accomplished via the glyoxylate shunt and TAC, and also as a result of action of pyruvate synthase and the production of C4- and C5-organic acids with the participation of CO2. The pathways of acetate metabolism depend on the growth conditions and on the presence of sulphide in the medium.

Acetates↗
PubMed1993

Biogenesis of the hydrogenosome in the anaerobic protist Trichomonas vaginalis.

Trichomonas vaginalis is a primitive protist that lacks mitochondria and peroxisomes. This aerotolerant anaerobe does, however, contain a specialized organelle involved in carbohydrate metabolism called the hydrogenosome. Hydrogenosomes are the site of fermentative oxidation of pyruvate, coupled to ATP production via substrate level phosphorylation. The organelle is surrounded by 2 membranes and appears to contain no genetic material. Hydrogenosomes are proposed to have arisen either through the conversion of mitochondria or via endosymbiosis with an anaerobic bacterium. Our studies show that hydrogenosomal proteins are synthesized on free polyribosomes and are, thus, posttranslationally translocated into the organelle. The 2 hydrogenosomal proteins we have examined in detail, ferredoxin and the beta subunit of succinyl CoA synthetase, appear to be synthesized as larger precursors that contain an 8- or 9-amino acid amino-terminal extension, respectively. These apparent leader sequences are absent from the mature proteins found in hydrogenosomes. The biochemical properties of the amino-terminal extensions are similar to one another and to mitochondrial presequences. These observations are consistent with the hypotheses that the specificity of protein translocation into hydrogenosomes is conferred by a short amino-terminal signal sequence and that the mechanisms underlying translocation are similar to that of mitochondria. Using in vitro import assays, we have shown that this short leader sequence is absolutely required for association of hydrogenosomal proteins with the organelle and that this association is dependent on ATP, temperature, and the presence of intact organelles.

Adenosine Triphosphate↗
PubMed2002

Effect of aniA (carbon flux regulator) and PhaC (poly-beta-hydroxybutyrate synthase) mutations on pyruvate metabolism in Rhizobium etli.

The Rhizobium etli poly-beta-hydroxybutyrate synthase (PhaC) mutant SAM100 grows poorly with pyruvate as the carbon source. The inactivation of aniA, encoding a global carbon flux regulator, in SAM100 restores growth of the resulting double mutant (VEM58) on pyruvate. Pyruvate carboxylase (PYC) activity, pyc gene transcription, and holoenzyme content, which were low in SAM100, were restored in strain VEM58. The genetically engineered overexpression of PYC in SAM100 also allowed its growth on pyruvate. The possible relation between AniA, pyc transcription, and reduced-nucleotide levels is discussed.

Acyltransferases↗
PubMed1989

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit.

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.

Acetolactate Synthase↗
PubMed1981

Independent control of selected insulin-sensitive cell membrane and intracellular functions-the linkage of cell membrane and intracellular events controlled by insulin. III. The influence of trypsin on cell membrane hexose transport and on glycogen synthase and mitochondrial pyruvate dehydrogenase activation.

Brief treatment of rat adipocytes with low concentration of trypsin activated both cell membrane and intracellular insulin-sensitive functions in marked contrast H2O2 (1), increase in pH, and oxidized glutathione (papers I and II). Glucose oxidation was activated maximally by trypsin in 30 s and preceded maximal activation of glycogen synthase, which occurred in 60s. Trypsin action to activate glycogen synthase was further enhanced by insulin. Mitochondrial pyruvate dehydrogenase was also rapidly activated by trypsin. With both insulin and trypsin action, mediator generation was directly demonstrated by glycogen synthase phosphoprotein phosphatase activation. Trypsin is thus the most insulin-like of these four agents studied since it acts by the formation of chemical mediator peptide(s) which are similar but not identical to those produced by insulin.

Adipose Tissue↗
PubMedDate not supplied

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate.

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.

Escherichia coli↗
PubMed2006

Disruption of the operon encoding Ehb hydrogenase limits anabolic CO2 assimilation in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.

Acetate-CoA Ligase↗
PubMed1983

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase.

In this paper, physicochemical evidence is given for the association between the pyruvate dehydrogenase complex (EC 1.2.4.1) and citrate synthase (EC 4.1.3.7) with two gel chromatographic techniques with poly(ethylene glycol) co-precipitation and with ultracentrifugation. Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state. Citrate synthase binds to the isolated transacetylase core of pyruvate dehydrogenase complex, but in the binding to the whole pyruvate dehydrogenase complex the two other components of the complex are also involved. One pyruvate dehydrogenase complex can bind 10-11 citrate synthase dimers, and the dissociation constant is about 5.7-6.0 microM as determined by two independent methods. The association between the pyruvate dehydrogenase complex and citrate synthase raises the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetyl-CoA from pyruvate towards citrate.

Animals↗
PubMed2006

Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate.

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.

Amino Acid Sequence↗
PubMed1998

Autoantibodies against subunits of pyruvate dehydrogenase and citrate synthase in a case of paediatric biliary cirrhosis.

In a newborn girl with a history of connatal liver damage, histological examination of a liver biopsy sample taken during the seventh week of life revealed incipient destruction of bile ducts. Very high titres of antimitochondrial antibodies were later detected in the plasma. As the hepatic injury tended towards fibrosis, the histological diagnosis became primary biliary cirrhosis. Autoantibodies against E1 alpha, E2, and E3 subunits and protein X component of pyruvate dehydrogenase complex, and against citrate synthase were detected on western immunoblotting in a 1 in 1000 dilution of the patient's serum. The patient died of her illness at 11 years of age. In liver specimens obtained at autopsy human immunoglobulin deposition was detected on the surface of almost all hepatic cells by immunohistology. As there is a physical and functional interaction between pyruvate dehydrogenase and citrate synthase within the mitochondria, the presence of autoantibodies against certain proteins in the patient suggests that in this form of the disease the molecular recognition and then the autoimmunisation process could be directed against a mitochondrial enzyme cluster containing both pyruvate dehydrogenase and citrate synthase.

Autoantibodies↗