Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.
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Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya. (+info)
Carbon-13 nuclear magnetic resonance study of metabolism of propionate by Escherichia coli.
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We have evaluated the use of [1,2-13C2]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13C-13C-12C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli. Additionally, formation of 13C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2-13C2]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1-13C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1, 2-13C2]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis. (+info)
Short-chain acyl-CoA-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a plant pathogen which causes grain rot and seedling rot of rice via the oxalate production.
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In Burkholderia glumae (formerly named Pseudomonas glumae), isolated as the causal agent of grain rot and seedling rot of rice, oxalate was produced from oxaloacetate in the presence of short-chain acyl-CoA such as acetyl-CoA and propionyl-CoA. Upon purification, the enzyme responsible was separated into two fractions (tentatively named fractions II and III), both of which were required for the acyl-CoA-dependent production of oxalate. In conjugation with the oxalate production from oxaloacetate catalyzed by fractions II and III, acetyl-CoA used as the acyl-CoA substrate was consumed and equivalent amounts of CoASH and acetoacetate were formed. The isotope incorporation pattern indicated that the two carbon atoms of oxalate are both derived from oxaloacetate, and among the four carbon atoms of acetoacetate two are from oxaloacetate and two from acetyl-CoA. When the reaction was carried out with fraction II alone, a decrease in acetyl-CoA and an equivalent level of net utilization of oxaloacetate were observed without appreciable formation of CoASH, acetoacetate or oxalate. It appears that in the oxalate production from oxaloacetate and acetyl-CoA, fraction II catalyzes condensation of the two substrates to form an intermediate which is split into oxalate and acetoacetate by fraction III being accompanied by the release of CoASH. (+info)
Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate.
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Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase. (+info)
Carbon metabolism in developing soybean root nodules: the role of carbonic anhydrase.
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A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development. (+info)
Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis.
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Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose (13)C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117-26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2, 3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter. (+info)
Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose.
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Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By (13)C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol. (mg of protein)(-1). min(-1), respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol. [mg of protein](-1). min(-1)) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol. [mg of protein](-1). min(-1)). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol. [mg of protein](-1). min(-1)) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures. (+info)
Another unusual type of citric acid cycle enzyme in Helicobacter pylori: the malate:quinone oxidoreductase.
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The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps. (+info)