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Query: EC:6.4.1.1 (pyruvate carboxylase)
 1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gluconeogenic capacity may be an important factor regulating dry matter intake (DMI) in lactating dairy cows. To determine whether increased glucose demand affects feed intake and hepatic gene expression, lactating Holstein cows were treated with phlorizin or vehicle (propylene glycol) for 7 d. Multiparous cows (n = 12; 269 +/- 65 d in milk, mean +/- SD) were randomly assigned to treatment sequence in a crossover design and were adapted to a common diet for 7 d before the beginning of the experiment. Phlorizin injected s.c. at 4 g/d caused glucose excretion in urine at the rate of 474 g/d. Although phlorizin decreased lactose synthesis and milk production (both P < 0.01), DMI and 3.5% fat-corrected milk production were not altered by treatment. A net deficit of 383 g glucose/d in milk and urine for phlorizin (relative to control) was likely replaced partially through increased gluconeogenesis. The molar insulin:glucagon ratio was decreased 17% by phlorizin (P < 0.001) and hepatic phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and pyruvate carboxylase mRNA abundance increased (all P < 0.05). Late-lactation dairy cows adapted quickly to an increase in peripheral glucose demand; adaptation mechanisms likely included enhanced gluconeogenic capacity, whereas DMI was not altered.
J Nutr 2005 Sep
PMID:Phlorizin administration increases hepatic gluconeogenic enzyme mRNA abundance but not feed intake in late-lactation dairy cows. 1614 Aug 99

We demonstrate a facile blue native polyacrylamide gel electrophoresis (BN-PAGE) technique to detect two malate-generating enzymes, namely fumarase (FUM), malate synthase (MS) and four oxaloacetate-forming enzymes, namely pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), citrate lyase (CL) and aspartate aminotransferase (AST). Malate dehydrogenase (MDH) was utilized as a coupling enzyme to detect either malate or oxaloacetate in the presence of their respective substrates and cofactors. The latter four oxaloacetate-forming enzymes were identified by 2,6-dichloroindophenol (DCIP) and p-iodonitrotetrazolium (INT) while the former two malate-producing enzymes were visualized by INT and phenazine methosulfate (PMS) in the reaction mixtures, respectively. The band formed at the site of enzymatic activity was easily quantified, while Coomassie staining provided information on the protein concentration. Hence, the expression and the activity of these enzymes can be readily evaluated. A two-dimensional (2D) BN-PAGE or SDS-PAGE enabled the rapid purification of the enzyme of interest. This technique also provides a quick and inexpensive means of quantifying these enzymatic activities in normal and stressed biological systems.
J Biochem Biophys Methods 2005 Sep 30
PMID:Blue native polyacrylamide gel electrophoresis and the monitoring of malate- and oxaloacetate-producing enzymes. 1615 36

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway.
Appl Microbiol Biotechnol 2007 Sep
PMID:Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum. 1733 67

To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines, FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the three clones was decreased by 21-39%, which was because of a decreased specific glucose uptake rate and yield of lactate from glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO cell culture can be efficiently reduced.
Appl Microbiol Biotechnol 2007 Sep
PMID:Functional expression of human pyruvate carboxylase for reduced lactic acid formation of Chinese hamster ovary cells (DG44). 1758 7

The transcriptional regulator CcpN of Bacillus subtilis has been recently characterized as a repressor of two gluconeogenic genes, gapB and pckA, and of a small noncoding regulatory RNA, sr1, involved in arginine catabolism. Deletion of ccpN impairs growth on glucose and strongly alters the distribution of intracellular fluxes, rerouting the main glucose catabolism from glycolysis to the pentose phosphate (PP) pathway. Using transcriptome analysis, we show that during growth on glucose, gapB and pckA are the only protein-coding genes directly repressed by CcpN. By quantifying intracellular fluxes in deletion mutants, we demonstrate that derepression of pckA under glycolytic condition causes the growth defect observed in the ccpN mutant due to extensive futile cycling through the pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate kinase. Beyond ATP dissipation via this cycle, PckA activity causes a drain on tricarboxylic acid cycle intermediates, which we show to be the main reason for the reduced growth of a ccpN mutant. The high flux through the PP pathway in the ccpN mutant is modulated by the flux through the alternative glyceraldehyde-3-phosphate dehydrogenases, GapA and GapB. Strongly increased concentrations of intermediates in upper glycolysis indicate that GapB overexpression causes a metabolic jamming of this pathway and, consequently, increases the relative flux through the PP pathway. In contrast, derepression of sr1, the third known target of CcpN, plays only a marginal role in ccpN mutant phenotypes.
J Bacteriol 2008 Sep
PMID:CcpN controls central carbon fluxes in Bacillus subtilis. 1858 36

Escherichia coli strain DC1515, deficient in glucose phosphotransferase (ptsG), lactate dehydrogenase (ldhA) and pyruvate:formate lyase (pflA), is a promising candidate for the fermentative production of succinate. To further improve the succinate producing capability of DC1515, we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase (pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515. We used lactose as a substitute of IPTG to induce pyc. We optimized the culture conditions such as the lactose addition time, the lactose concentration and the culture temperature after induction for succinate production. We also explored the effect of lactose supplement during the fermentation. The results showed that pyc can be expressed under lactose induction in the fermentative medium with 15 g/L glucose due to the deficient of ptsG in DC1515. Under optimized conditions, the final succinate concentration reached to 15.17 g/L, which was 1.78-fold higher than that of control strain. If complementing lactose twice to the concentration of 1 g/L during the fermentation, the final succinate concentration could further reach to 17.54 g/L. This work might provide valuable information for gene expression in E. coli strains using lactose as inducer for succinate production in a glucose-medium. Due to the reduced cost, E. coli is becoming a more promising strain for succinate production through fermentation.
Sheng Wu Gong Cheng Xue Bao 2009 Sep
PMID:[Expression of heterogenous pyruvate carboxylase in Escherichia coli with lactose as inducer and its effect on succinate production]. 1993 76

Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional role, which is unrelated to the enzyme activity, represents an example of a special form of multifunctionality called moonlighting. We have performed a detailed site-directed mutagenesis approach to elucidate which region(s) of H. polymorpha pyruvate carboxylase are involved in its second function. This resulted in the identification of three amino acids that are essential for the moonlighting function. Mutating these residues in a single mutant protein fully inactivated the moonlighting function, but not the enzyme activity of pyruvate carboxylase because the strain was prototrophic. A 3D homology model revealed that all three residues are positioned at the side of a TIM barrel where the N-terminal ends of the beta-strands are located. This is a novel observation as the TIM barrel proteins invariably are enzymes and have their catalytic side at the C-terminal end of the beta-sheets. Our finding implies that a TIM barrel fold can also fulfill a non-enzymatic function and that this function can reside at the N-terminal end of the barrel.
Biochim Biophys Acta 2010 Sep
PMID:The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel. 2035 4

The present study aimed at investigating whether increasing concentrations of glucose supply have a depressive effect on the mRNA abundance and activity of key gluconeogenic enzymes in dairy cows. Twelve Holstein-Friesian dairy cows in mid-lactation were intravenously infused with saline (SI; n = 6) or a 40% glucose solution (GI; n = 6). For GI cows, the infusion dose increased by 1.25%/d relative to the initial NE(l) requirement until a maximum dose equating to surplus 30% NE(l) was reached on d 24. Cows receiving SI received an equivalent volume of 0.9% saline solution. Blood samples were taken every 2 d, and liver biopsies were collected every 8 d. A treatment x quadratic dose interaction (P < 0.01) was observed for the concentration of plasma glucose and serum insulin. The interactions were due to positive quadratic responses of the concentrations of glucose and insulin for GI cows, whereas the concentrations of glucose and insulin did not change over time for SI cows. The concentration of beta-hydroxybutyric acid (BHBA) and serum urea nitrogen (BUN) responded in a treatment x quadratic dose manner, such that greater decreases (P < 0.01) in BHBA and BUN concentrations were observed for cows receiving GI than SI as the dosage increased. Serum NEFA concentration tended to follow a similar pattern as serum BHBA and BUN; however, the interaction was not significant (P = 0.07). The mRNA abundance of gluconeogenesis enzymes followed a linear treatment x dose interaction (P < 0.05) for only pyruvate carboxylase (PC), which was paralleled by a trend for a linear treatment x dose interaction (P = 0.13) for PC enzyme activity. The least PC expression and activity were observed at the largest glucose dosage. The activity, but not mRNA abundance, of fructose 1,6-bisphosphatase (FBPase) showed treatment x quadratic dose interactions (P < 0.05) with decreasing activity at increasing glucose dose. Activities and expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase were not affected (P > 0.25) by treatment. In conclusion, hepatic gluconeogenic enzymes are only moderately affected by slowly increasing glucose supply, including a translational or posttranslational downregulation of FBPase activity and a decrease in the mRNA abundance of PC with possible consequences for PC enzyme activity.
J Anim Sci 2010 Sep
PMID:Expression and activity of key hepatic gluconeogenesis enzymes in response to increasing intravenous infusions of glucose in dairy cows. 2049 14

d-Amino acid oxidase (DAO) is an important flavo-enzyme that catalyzes the oxidative deamination of d-amino acids into the corresponding alpha-keto acid, ammonia and H(2)O(2). We identified two amino acid oxidases in the methylotrophic yeast Pichia pastoris: Dao1p, which preferentially uses d-alanine as a substrate, and Dao2p, which uses d-aspartate as a preferred substrate. Dao1p has a molecular mass of 38.2 kDa and a pH optimum of 9.6. This enzyme was localized to peroxisomes, albeit a typical peroxisomal targeting signal is lacking. Interestingly, P. pastoris mutant strains, defective in the enzyme pyruvate carboxylase, showed a pronounced growth defect on d-alanine, concomitant with a significant reduction in Dao1p activity relative to the wild-type control. This indicates that pyruvate carboxylase functions in import and/or activation of P. pastoris Dao1p.
FEMS Yeast Res 2010 Sep
PMID:Activation of a peroxisomal Pichia pastoris D-amino acid oxidase, which uses d-alanine as a preferred substrate, depends on pyruvate carboxylase. 2055 May 80

Genetic engineering of fluorescent pseudomonads for various industrially, agriculturally and environmentally important bioprocesses often involves the use of suitable plasmids. Plasmid-mediated alterations in host physiology and metabolism are poorly understood for this group of organisms. Thus, we investigated the metabolic perturbations in Pseudomonas fluorescens 13525 due to the independent and combined presence of broad-host-range plasmids, pBBR1MCS-2 (copy number 30) and pUCPM18 derived pAB4 and pAB8 (copy number 14-16). Presence of pAB4 and pAB8 not only significantly increased the growth rate and glucose utilization of P. fluorescens 13525, but also increased glucose dehydrogenase activity and gluconic acid production indicating enhanced direct oxidative pathway for glucose catabolism. Additionally, increased secretion of pyruvic, acetic, and citric acids caused faster media acidification in presence of pAB4 and pAB8. Simultaneous presence of pAB4/pAB8 in Pf (pAB48) and pAB4/pBBR1MCS-2 in Pf (pAB4BBR1MCS-2) reduced their respective copy numbers to nearly half. Pf (pAB48) demonstrated further increase in direct oxidation pathway without altering growth and glucose depletion rates, as compared with single transformants. Conversely, pBBR1MCS-2 plasmid did not greatly alter P. fluorescens 13525 metabolism when present independently but masked the effects imposed by pAB4 when present in its combination. In conclusion, P. fluorescens 13525 redesigns its metabolism in response to the presence of plasmids irrespective of their nature, by enhancing anaplerosis with a simultaneous reduction in catabolism as indicated by increased pyruvate carboxylase and decreased citrate synthase activities, respectively. Such information will be helpful for vector designing during genetic engineering of fluorescent pseudomonads.
Appl Microbiol Biotechnol 2010 Sep
PMID:Broad-host-range plasmid-mediated metabolic perturbations in Pseudomonas fluorescens 13525. 2057 95


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