Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.
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PMID:Pathway analysis and metabolic engineering in Corynebacterium glutamicum. 1107 21

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass isotopomers of choice remain the same. In special cases, reduced labeling degree of the tracer substrate can increase the precision of flux analysis. Enhanced precision and flux information can be achieved via multiply labeled substrates. The presented approach can be applied for effective experimental design of (13)C tracer studies for metabolic flux analysis. Intensity ratios of other products such as glutamate, valine, phenylalanine, and riboflavin also sensitively reflect flux parameters, which underlines the great potential of mass spectrometry for flux analysis.
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PMID:Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry. 1128 93

Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.
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PMID:Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. 1150 84

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.
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PMID:Metabolic flux responses to pyruvate kinase knockout in Escherichia coli. 1174 55

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.
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PMID:Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures. 1191 94

Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.
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PMID:Metabolic network analysis of Bacillus clausii on minimal and semirich medium using (13)C-labeled glucose. 1200 95

The changes in the intermediary metabolism of plant cells were quantified according to growth conditions at three different stages of the growth cycle of tomato cell suspension. Eighteen fluxes of central metabolism were calculated from (13)C enrichments after near steady-state labeling by a metabolic model similar to that described in Dieuaide-Noubhani et al. (Dieuaide-Noubhani, M., Raffard, G., Canioni, P., Pradet, A., and Raymond, P. (1995) J. Biol. Chem. 270, 13147-13159), and 10 net fluxes were obtained directly from end-product accumulation rates. The absolute flux values of central metabolic pathways gradually slowed down with the decrease of glucose influx into the cells. However, the relative fluxes of glycolysis, the pentose-P pathway, and the tricarboxylic acid cycle remained unchanged during the culture cycle at 70, 28, and 40% of glucose influx, respectively, and the futile cycle of sucrose remained high at about 6-fold the glucose influx, independently from carbon nutritional conditions. This natural resistance to flux alterations is referred to as metabolic stability. The numerous anabolic pathways, including starch synthesis, hexose accumulation, biosynthesis of wall polysaccharides, and amino and organic acid biosynthesis were comparatively low and variable. The phosphoenolpyruvate carboxylase flux decreased 5-fold in absolute terms and 2-fold in relation to the glucose influx rate during the culture cycle. We conclude that anabolic fluxes constitute the flexible part of plant cell metabolism that can fluctuate in relation to cell demands for growth.
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PMID:The metabolic architecture of plant cells. Stability of central metabolism and flexibility of anabolic pathways during the growth cycle of tomato cells. 1222 84

To determine enzymatic activities in the thermotolerant strain K1 (formerly "Sulfobacillus thermosulfidooxidans subsp. thermotolerans"), it was grown in a mineral medium with (1) thiosulfate and Fe2+ or pyrite (autotrophic conditions), (2) Fe2+, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways. An increased content of carbon dioxide (up to 5 vol%) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide was fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO2 in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase decreased with the increasing content of CO2 in the medium.
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PMID:[The enzyme of carbon metabolism in the thermotolerant sulfobacillus strain K1]. 1252 95

In cold-hardened leaves (CHL) of winter rye (Secale cereale L.) much higher levels of malate were detected by (13)C-NMR than in non-hardened leaves (NHL). As this was not observed previously, malate metabolism of CHL was studied in more detail by biochemical assays. The activities of several enzymes of malate metabolism, NADP-malate dehydrogenase, NAD-malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-malic enzyme, were also increased in CHL. Short exposures to low temperature of 1-3 d did not induce increases in the malate content or in the activities of enzymes of malate metabolism in mature NHL. The malate content and the enzyme activities declined within 1-2 d after a transfer of CHL from their growing temperature of 4 degrees C to 22 degrees C. The malate content was further increased when CHL were exposed to a higher light intensity at 4 degrees C. In CO(2)-free air the malate content of CHL strongly declined at 4 degrees C. Malate may thus serve as an additional carbon sink and as a CO(2)-store in CHL. It may further function as a vacuolar osmolyte balancing increased concentrations of soluble sugars previously observed in the cytosol of CHL. Malate was not used as a source of reductants when CHL were exposed to photo-oxidative stress by treatment with paraquat. However, the activities of enzymes of the oxidative pentose phosphate pathway were markedly increased in CHL and may serve as non-photosynthetic sources of NADPH and thus contribute to the previously observed superior capacity of CHL of winter rye to maintain their antioxidants in a reduced state in the presence of paraquat.
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PMID:Malate metabolism and reactions of oxidoreduction in cold-hardened winter rye (Secale cereale L.) leaves. 1259 77

Using the carbon isotope labeling technique, the response of cyanobacterial central carbon metabolism to the change in environmental conditions was investigated. Synechocystis was grown in the heterotrophic and mixotrophic cultures fed with 13C-labeled glucose. The labeling patterns of the amino acids in biomass hydrolysates for both cultures were detected by the two-dimensional 1H-13C correlation nuclear magnetic resonance (2D 1H-13C COSY NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) technique. The in vivo intracellular flux distributions were then quantitated from the labeling measurements and metabolite balances using a parameters fitting approach. From the estimated flux distributions, it was found that the pentose phosphate pathway was the major pathway of glucose catabolism in the heterotrophic culture, while in the mixotrophic culture, the flux of CO2 fixation through the Calvin cycle was about two-fold of the glucose input flux. The relative flux through the phosphoenolpyruvate carboxylase was very high in both cultures, and this reaction represented about 25% of the assimilated CO2 in the mixotrophic culture. More importantly, we found a substantial outflow from the tricarboxylic acid cycle to glycolysis pathway carried by the malic enzyme, demonstrating the operation of a C4 pathway in cyanobacterial cells through the PEP carboxylase and malic enzyme. The estimated flux distributions also revealed that the NADPH synthesis was in excess relative to its requirement, and the excess NADPH might be reoxidized in cyanobacterial respiration to provide the energy for cellular requirement. Moreover, the analyzed result also suggested that the activity of the respiratory electron transport chain in cyanobacterial cells was not inhibited by light.
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PMID:Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose. 1261 90


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