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Regulation of pyruvate carboxylase in Rhizobium etli 总被引:1,自引:0,他引:1
Michael F Dunn Gisela Araza Miguel A Cevallos Jaime Mora 《FEMS microbiology letters》1997,157(2):301-306
3.
Delaunay S Daran-Lapujade P Engasser JM Goergen JL 《Journal of industrial microbiology & biotechnology》2004,31(4):183-188
The glutamate-producing bacterium, Corynebacterium glutamicum is known to possess two anaplerotic enzymes: pyruvate carboxylase (Pc) and phosphoenolpyruvate carboxylase (PEPc). In vitro, this latter enzyme appeared to be inhibited by different glutamic acid salts, whereas ammonium-glutamate had no influence on Pc activity. To investigate the in vivo relevance of PEPc activity inhibition, the intracellular concentration of glutamate was determined throughout the glutamate-producing process. The intracellular concentration was then shown to be sufficient to induce a dramatic inhibition of PEPc activity during the process. As a consequence, intracellular accumulation of glutamate could be at least partially responsible for the weak participation of PEPc within the anaplerosis activity in amino-acid-producing strains of C. glutamicum. 相似文献
4.
Abstract: The activity of the pyruvate carboxylase was determined in brains of newborn and adult mice as well as primary cultures of astrocytes, of cerebral cortex neurons, and of cerebellar granule cells. The activity was found to be 0.25 ± 0.14, 1.24 ± 0.07, and 1.75 ± 0.13 nmol · min−1 · mg−1 protein in, respectively, neonatal brain, adult brain, and astrocytes. Neither of the two types of neurons showed any detectable enzyme activity (i.e., < 0.05 nmol · min−1 · mg−1 ). It is therefore concluded that pyruvate carboxylase is an astrocytic enzyme. 相似文献
5.
Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum
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The physiological behavior of Corynebacterium glutamicum in glucose-limited chemostat cultures was examined from both growth kinetics and enzymatic viewpoints. Metabolic fluxes within the central metabolism were calculated from growth kinetics and analyzed in relation to specific enzyme activities. At high growth rates, incomplete glucose removal was observed, and this was attributed to rate-limiting capacity of the phosphotransferase system transporter and the probable contribution of a low-affinity permease uptake mechanism. The improved biomass yield observed at high growth rates was related to a shift in the profile of anaplerotic carboxylation reactions, with pyruvate carboxylase replacing malic enzyme. Phosphoenolpyruvate carboxylase, an activity often assumed to be the major anaplerotic reaction during growth of C. glutamicum on glucose, was present at only low levels and is unlikely to contribute significantly to tricarboxylic acid cycle fuelling other than at low growth rates. 相似文献
6.
<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> tailored for high-yield L-valine production 总被引:1,自引:0,他引:1
Blombach B Schreiner ME Bartek T Oldiges M Eikmanns BJ 《Applied microbiology and biotechnology》2008,79(3):471-479
We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L: -valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested. 相似文献
7.
A. Yu. Skorokhodova A. A. Stasenko A. Yu. Gulevich V. G. Debabov 《Applied Biochemistry and Microbiology》2018,54(2):141-148
The effect of anaplerotic pathways activation on CO2-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO2. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO2 formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO2 source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO2-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle. 相似文献
8.
Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes 总被引:5,自引:4,他引:1
William C. Gamberino Deborah A. Berkich Christopher J. Lynch Baiyang Xu Kathryn F. LaNoue 《Journal of neurochemistry》1997,69(6):2312-2325
Abstract: CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis. 相似文献
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Quantitative physiological characterization and isotopic tracer experiments revealed that pyruvate kinase mutants of Bacillus subtilis produced significantly more CO(2) from glucose in the tricarboxylic acid cycle than is explained by the remaining conversion of phosphoenolpyruvate (PEP) to pyruvate catalyzed by the phosphotransferase system. We show here that this additional catabolic flux into the tricarboxylic acid cycle was catalyzed by the PEP carboxykinase. In contrast to its normal role in gluconeogenesis, PEP carboxykinase can operate in the reverse direction from PEP to oxaloacetate upon knockout of pyruvate kinase in a riboflavin-producing B. subtilis strain and in wild-type 168. At least in the industrial strain, we demonstrate the additional capacity of PEP carboxykinase to function as a substitute anaplerotic reaction when the normal pyruvate carboxylase is inactivated. Presumably as a consequence of the unfavorable kinetics of an ATP-synthesizing anaplerotic PEP carboxykinase reaction, such pyruvate carboxylase mutants grow slowly or, as in the case of wild-type 168, not at all. 相似文献
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The compartmentation of amino acid metabolism is an active and important area of brain research. 13C labeling and 13C nuclear magnetic resonance (NMR) are powerful tools for studying metabolic pathways, because information about the metabolic histories of metabolites can be determined from the appearance and position of the label in products. We have used 13C labeling and 13C NMR in order to investigate the metabolic history of gamma-aminobutyric acid (GABA) and glutamate in rat brain. [1-13C]Glucose was infused into anesthetized rats and the 13C labeling patterns in GABA and glutamate examined in brain tissue extracts obtained at various times after infusion of the label. Five minutes after infusion, most of the 13C label in glutamate appeared at the C4 position; at later times, label was also present at C2 and C3. This 13C labeling pattern occurs when [1-13C]glucose is metabolized to pyruvate by glycolysis and enters the pool of tricarboxylic acid (TCA) intermediates via pyruvate dehydrogenase. The label exchanges into glutamate from the TCA cycle pool through glutamate transaminases or dehydrogenase. After 30 min of infusion, approximately 10% of the total 13C in brain extracts appeared in GABA, primarily (greater than 80%) at the amino carbon (C4), indicating that the GABA detected is labeled through pyruvate carboxylase. The different labeling patterns observed for glutamate and GABA show that the large detectable glutamate pool does not serve as the precursor to GABA. Our NMR data support previous experiments suggesting compartmentation of metabolism in brain, and further demonstrate that GABA is formed from a pool of TCA cycle intermediates derived from an anaplerotic pathway involving pyruvate carboxylase. 相似文献
12.
Cline GW Lepine RL Papas KK Kibbey RG Shulman GI 《The Journal of biological chemistry》2004,279(43):44370-44375
Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process. 相似文献
13.
Wenjuan Yao Xiaozhao Deng Hui Zhong Miao Liu Pu Zheng Zhihao Sun Yun Zhang 《Journal of industrial microbiology & biotechnology》2009,36(7):911-921
Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis.
The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC
13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by
the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in
ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production
strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis. 相似文献
14.
Teixeira AP Santos SS Carinhas N Oliveira R Alves PM 《Neurochemistry international》2008,52(3):478-486
In this work, brain cell metabolism was investigated by (13)C NMR spectroscopy and metabolic flux analysis (MFA). Monotypic cultures of astrocytes were incubated with labeled glucose for 38 h, and the distribution of the label was analyzed by (13)C NMR spectroscopy. The analysis of the spectra reveals two distinct physiological states characterized by different ratios of pyruvate carboxylase to pyruvate dehydrogenase activities (PC/PDH). Intracellular flux distributions for both metabolic states were estimated by MFA using the isotopic information and extracellular rate measurements as constraints. The model was subsequently checked with the consistency index method. From a biological point of view, the occurrence of the two physiological states appears to be correlated with the presence or absence of extracellular glutamate. Concerning the model, it can be stated that the metabolic network and the set of constraints adopted provide a consistent and robust characterization of the astrocytic metabolism, allowing for the calculation of central intracellular fluxes such as pyruvate recycling, the anaplerotic flux mediated by pyruvate carboxylase, and the glutamine formation through glutamine synthetase. 相似文献
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Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition. 相似文献
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Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a competitive inhibitor of chicken liver pyruvate carboxylase with respect to ATP. The chicken liver enzyme is unable to utilise FTP as a substrate at a measureable rate, but FTP is a poor substrate for the sheep liver enzyme. When FTP binds to the enzyme, its fluorescence is enhanced and in this way the formation of enzyme-FTP complexes can be monitored. Using this property of FTP, the effect of Mg2+ and acetyl-CoA on the binding of nucleoside triphosphates to the chicken liver enzyme was examined. Mg2+ was found to enhance the binding of FTP whilst acetyl-CoA reduced the fluorescence intensity of a mixture of Mg2+, enzyme and FTP. Most probably, this was caused by a conformational change in the enzyme which changed the environment of the fluorophore. 相似文献
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Immunocytochemical Examination of Neural Rat and Mouse Primary Cultures Using Monoclonal Antibodies Raised Against Pyruvate Carboxylase 总被引:5,自引:3,他引:2
Abstract: Pyruvate carboxylase (EC 6.4.1.1; PC) catalyzes the formation of oxaloacetate by energy-dependent fixation of CO2 to pyruvate. The aim of the present work was to generate antibodies against PC and use them to localize PC in the cells of astroglia-rich and neuron-rich primary cultures derived from the brains of rats and mice. Mouse monoclonal antibodies raised against the enzyme were shown to be monospecific as indicated by immunoblotting. The staining of the cells for PC appeared in grains. These represent mitochondria, as PC is known as a mitochondrial enzyme. Immunocytochemical examination of astroglia-rich primary cultures of rat or mouse brain cells revealed a colocalization of PC with the astroglial marker glial fibrillary acidic protein (GFAP) in many cells. However, there were GFAP-positive cells showing no specific staining for PC, and vice versa. Also, in neuron-rich primary cultures PC was found only in the ∼10% GFAP-expressing astroglial cells contaminating the neuron-rich primary culture, whereas it was absent from the neurons identified by antibodies against neuron-specific enolase. These results suggest that PC is predominantly an astroglial enzyme and that astroglial cells play an important role in the intermediary and the energy metabolism of the brain. 相似文献
18.
M E Walker D L Val M Rohde R J Devenish J C Wallace 《Biochemical and biophysical research communications》1991,176(3):1210-1217
In Saccharomyces cerevisiae, pyruvate carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from pyruvate. We report here the existence of two pyruvate carboxylase isozymes, which are encoded by separate genes within the yeast genome. Null mutants were constructed by one step gene disruption of the characterised PYC gene in the yeast genome. The mutants were found to have 10-20% residual pyruvate carboxylase activity, which was attributable to a protein of identical size and immunogenically related to pyruvate carboxylase. Immunocytochemical labelling studies on ultrathin sections of embedded whole cells from the null mutants showed the isozyme to be located exclusively in the cytoplasm. We have mapped the genes encoding both enzymes and shown the previously characterised gene, designated PYC1, to be on chromosome VII whilst PYC2 is on chromosome II. 相似文献
19.
Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, beta-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase. 相似文献
20.
Biotin-dependent enzymes catalyze carboxyl transfer reactions by efficiently coordinating multiple reactions between spatially distinct active sites. Pyruvate carboxylase (PC), a multifunctional biotin-dependent enzyme, catalyzes the bicarbonate- and MgATP-dependent carboxylation of pyruvate to oxaloacetate, an important anaplerotic reaction in mammalian tissues. To complete the overall reaction, the tethered biotin prosthetic group must first gain access to the biotin carboxylase domain and become carboxylated and then translocate to the carboxyltransferase domain, where the carboxyl group is transferred from biotin to pyruvate. Here, we report structural and kinetic evidence for the formation of a substrate-induced biotin binding pocket in the carboxyltransferase domain of PC from Rhizobium etli. Structures of the carboxyltransferase domain reveal that R. etli PC occupies a symmetrical conformation in the absence of the biotin carboxylase domain and that the carboxyltransferase domain active site is conformationally rearranged upon pyruvate binding. This conformational change is stabilized by the interaction of the conserved residues Asp590 and Tyr628 and results in the formation of the biotin binding pocket. Site-directed mutations at these residues reduce the rate of biotin-dependent reactions but have no effect on the rate of biotin-independent oxaloacetate decarboxylation. Given the conservation with carboxyltransferase domains in oxaloacetate decarboxylase and transcarboxylase, the structure-based mechanism described for PC may be applicable to the larger family of biotin-dependent enzymes. 相似文献