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At least 649 records · Page 36Linked to original sources
PubMed2002

Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation.

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD(+). Expression of low-K(m) Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.

Citrate (si)-Synthase↗
PubMed2001

Transcriptional regulation of phosphoenolpyruvate synthase by maltose in the hyperthermophilic archaeon, Pyrococcus furiosus.

The phosphoenolpyruvate synthase (EC 2.7.9.2) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the Pi-dependent formation of pyruvate, ATP and water from phosphoenolpyruvate and AMP [Sakuraba, H., Arch. Biochem. Biophys., 364, 125-128 (1999)]. In this study, the P. furiosus phosphoenolpyruvate synthase was purified to homogeneity and the N-terminal amino acid sequence was determined to be AYRFIKWFEELS. The sequence coincided completely with the N-terminal amino acid sequence of the translation product of the mlrA gene that was found to be upregulated at the transcriptional level by the alpha-linked glucose disaccharide maltose [Robinson, K.A. and Schreier, H.J., Gene, 151, 173-176 (1994)]. The mlrA gene was cloned and expressed in Escherichia coli. The recombinant cells produced a hyperthermostable phosphoenolpyruvate synthase. This indicates that the mlrA gene encodes the enzyme. When P. furiosus was grown on the medium supplemented with maltose, the specific activity of the enzyme markedly increased (about 10-fold) compared with that produced by the cells grown on the medium without maltose. Northern blot analysis revealed enhanced transcription of the mlrA gene in the presence of maltose. These results indicate that transcriptional regulation of phosphoenolpyruvate synthase by maltose is present in P. furiosus.

Journal Article↗
PubMed2002

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen.

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.

Acetaldehyde↗
PubMed2005

Assessment of a dual regulatory role for NO in liver regeneration after partial hepatectomy: protection against apoptosis and retardation of hepatocyte proliferation.

The role of hepatic nitric oxide (NO) in liver regeneration after partial hepatectomy (PH) was studied in animals carrying a nitric oxide synthase-2 transgene under the control of the phospho(enol)pyruvate carboxykinase promoter. These mice expressed NOS-2 in liver cells under fasting conditions. Liver mass recovery and molecular parameters related to cell proliferation were determined after PH. Preexisting hepatic NO synthesis, as well as NO delivery by NO-donors, impaired early signaling (for example, attenuated NF-kappaB activation and TNF-alpha and IL-6 release). The regenerative process was also impaired as a result of an insufficient proliferative response, but mouse survival after surgery was not compromised. However, NO exerted a protective role against apoptosis in transgenic hepatectomized mice. Local production of NO in liver cells, achieved by hydrodynamic-based transfection with a NOS-2-encoding plasmid, also resulted in delayed liver recovery after PH and also protected against Fas-mediated apoptosis. These data show that sustained presence of NO after PH exerts a dual role: attenuating liver regeneration while efficiently protecting against liver apoptosis.

Animals↗
PubMed1989

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme.

We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and alpha-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagensis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.

Acetates↗
PubMed1990

Studies on the metabolism of glycolyl-CoA.

Glycolyl-CoA can be formed during the course of the beta-oxidation by rat liver mitochondria of 4-hydroxybutyrate. The existence of this beta-oxidation has been previously supported by the occurrence of 4-hydroxybutyrate and its beta-oxidation catabolites in urine from patients with 4-hydroxybutyric aciduria, an inborn error of gamma-aminobutyric acid metabolism due to the deficiency of succinic semialdehyde dehydrogenase. The characteristics of the mitochondrial beta-oxidation of 4-hydroxybutyrate were, in rat liver, compared with those of the mitochondrial beta-oxidation of butyrate. The inhibition by malonate of the oxidation of 4-hydroxybutyrate was about twofold weaker than that of oxidation of butyrate, whereas both oxidations were abolished by preincubating the mitochondria with 1 mM valproic acid, a known inhibitor of mitochondrial beta-oxidation. Mitochondria from rat kidney cortex were demonstrated to catalyse, as previously shown for hepatic mitochondria, the carnitine-dependent oxidation of 12-hydroxylauroyl-CoA-omega-Hydroxymonocarboxylyl-CoAs are thus concluded to be precursors of glycolyl-CoA also in rat kidney cortex. In addition, 3-hydroxypyruvate was found to be a precursor of glycolyl-CoA, since it was oxidized by bovine heart pyruvate dehydrogenase with a cofactor requirement similar to that of pyruvate oxidation. Glycolyl-CoA was a substrate of carnitine acetyltransferase (pigeon breast muscle). Pig heart citrate synthase was capable of catalyzing the condensation of glycolyl-CoA with oxaloacetate. The product of this reaction induced low NADH production rates dependent on the addition of porcine heart aconitase and isocitrate dehydrogenase.

Acetate-CoA Ligase↗
PubMed2005

C-C bond formation and cleavage in radical enzymes, a theoretical perspective.

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C-C bond formation or cleavage reactions.

Acetyltransferases↗
PubMed1997

Metabolic regulation in mammalian hibernation: enzyme and protein adaptations.

Mammalian hibernation requires specific regulatory controls on metabolism to coordinate entry, maintenance, and arousal stages, as well as adjustments to many metabolic functions to support long-term dormancy. Several mechanisms of metabolic regulation are involved in potentiating survival. One of these is the reversible phosphorylation of regulatory enzymes, including glycogen phosphorylase, phosphofructokinase, pyruvate kinase, and pyruvate dehydrogenase. In particular, the sharp suppression of pyruvate dehydrogenase during hibernation shows the importance of control over mitochondrial oxidative metabolism for reducing metabolic rate. Fine control over specific enzymes also occurs via differential temperature effects on kinetic and allosteric properties. Analysis of temperature effects on the properties of pyruvate kinase, fructose-1,6-bisphosphatase, creatine kinase, and citrate synthase from ground squirrel or bat tissues shows a range of responses, some that would reduce enzyme activity in the hibernating state and some that would promote temperature-insensitive enzyme function. Reduced tissue phosphagen and adenylate levels, but not energy charge, may also contribute to overall metabolic suppression. New research is exploring the role of transcriptional and translational controls in hibernation via several approaches. For example, immunoblotting with antibodies to heat shock proteins (hsp 70 family) revealed the presence of constitutive hsc 70 in bat tissues but levels of the protein did not change between euthermic and hibernating states and neither the inducible hsp 70 nor the glucose-responsive protein grp 78 appeared during hibernation.

Adaptation, Physiological↗
PubMed2001

Effect of sesame seeds rich in sesamin and sesamolin on fatty acid oxidation in rat liver.

Activities of enzymes involved in hepatic fatty acid oxidation and synthesis among rats fed sesame (Sesamum indicum L.) differing in lignan content (sesamin and sesamolin) were compared. Sesame seeds rich in lignans from two lines, 0730 and 0732, lines established in this laborary, and those from a conventional cultivar (Masekin) were employed. Seeds from the 0730 and 0732 lines contained sesamin and sesamolin at amounts twice those from Masekin. Sesame seeds were added at levels of 200 g/kg to the experimental diets. Sesame increased both the hepatic mitochondrial and the peroxisomal fatty acid oxidation rate. Increases were greater with sesame rich in lignans than with Maskin. Noticeably, peroxisomal activity levels were >3 times higher in rats fed diets containing sesame seeds from the 0730 and 0732 lines than in those fed a control diet without sesame. The diet containing Masekin seed caused only a 50% increase in the value, however. Diets containing seeds from the 0730 and 0732 lines, compared to the control and Masekin diets, also significantly increased the activity of hepatic fatty acid oxidation enzymes including acyl-CoA oxidase, carnitine palmitoyltranferase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase. In contrast, diets containing sesame lowered the activity of enzymes involved in fatty acid synthesis including fatty acid synthase, glucose-6-phosphate dehydrogenase, ATP-citrate lyase, and pyruvate kinase. No significant differences in enzyme activities were, however, seen among diets containing sesame from Masekin cultivar and lines 0730 and 0732. Serum triacylglycerol concentrations were lower in rats fed diets containing sesame from lines 0730 and 0732 than in those fed the control or Masekin diet. It is apparent that sesame rich in lignans more profoundly affects hepatic fatty acid oxidation and serum triacylglycerol levels. Therefore, consumption of sesame rich in lignans results in physiological activity to alter lipid metabolism in a potentially beneficial manner.

Animals↗
PubMed2006

ChREBP*Mlx is the principal mediator of glucose-induced gene expression in the liver.

In mammals, glucose-regulated gene expression has been best characterized in the liver, where increased glucose metabolism induces transcription of genes encoding enzymes involved in de novo lipogenesis. ChREBP and Mlx dimerize and function together as a glucose-responsive transcription factor to regulate target genes, such as liver-type pyruvate kinase, acetyl-CoA carboxylase 1, and fatty acid synthase. To identify additional glucose-responsive genes in the liver, we used microarray analysis to compare gene expression patterns in low and high glucose conditions in hepatocytes. Target genes of ChREBP.Mlx were simultaneously identified by gene profiling in the presence or absence of a dominant negative Mlx. Of 224 genes that are induced by glucose, 139 genes (62%) were also inhibited by the dominant negative Mlx. Lipogenic enzyme genes involved in the entire pathway of de novo lipogenesis were found to be glucose-responsive target genes of ChREBP.Mlx. Genes encoding enzymes in other metabolic pathways and numerous regulators of metabolism were also identified. To determine if any of these genes are direct targets of ChREBP.Mlx, we searched for ChoRE-like sequences in the 5'-flanking regions of several genes that responded rapidly to glucose. ChoRE sequences that bound to ChREBP.Mlx and supported a glucose response were identified in two additional genes. Combining all of the known ChoRE sequences, we generated a modified ChoRE consensus sequence, CAYGNGN(5)CNCRTG. In summary, ChREBP.Mlx is the principal transcription factor regulating glucose-responsive genes in the liver and coordinately regulates a family of genes required for glucose utilization and energy storage.

Animals↗
PubMed1983

The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation.

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Aldose-Ketose Isomerases↗
PubMed1973

Catalytic studies on tryptophanase from Bacillus alvei.

Tryptophanase from Bacillus alvei exhibited the expected spectrum of pyridoxal-5'-phosphate-dependent reactions. It exhibited l-serine dehydratase, S-alkyl-cysteine lyase, and cysteine desulfhydrase activities, as well as the classic tryptophanase reactions (all beta elimination reactions). It also acted as a tryptophan synthetase (beta replacement reactions) using indole plus l-serine or l-cysteine or S-methyl-cysteine as substrates. The beta elimination reactions are simple competitors of the replacement reactions for the same amino acid substrates. The kinetics of the reactions were examined in detail using a coupled continuous spectrophotometric assay. A product (indole) inhibition study of the beta elimination reaction with tryptophan showed simple, noncompetitive inhibition; the same study with allosubstrates showed noncompetitive inhibition by indole. These product studies provided data on the beta replacement reactions as well. The results are discussed in terms of a mechanism for the B. alvei tryptophanase.

Bacillus↗
PubMed2003

Potential drug targets and progress towards pharmacologic inhibition of hepatic glucose production.

A number of therapeutic targets are currently under investigation for inhibition of hepatic glucose production with small molecules. Antagonists of the glucagon receptor, glycogen phosphorylase, 11-beta-hydroxysteroid dehydrogenase-1 and fructose 1,6-bisphosphatase are, or have been, under evaluation in human clinical trials. Other strategies, including glucocorticoid receptor antagonists and carnitine palmitoyltransferase inhibitors, are supported by proof of principle studies in man as well as rodents. Several potential targets including glucose-6-phosphatase, glucose-6-phosphatase translocase, glycogen synthase kinase-3, adenosine receptor 2B antagonists, phosphoenolpyruvate carboxykinase and pyruvate dehydrogenase kinase, have been validated by compounds that are effective in animal models. Other targets like PGC-1a and CREB have initial validation support but no medicinal chemistry has been reported.

Animals↗