Home About us Contact
|
Home About us Contact | ||
|
|
||
Tricarboxylic Acid (tricarboxylic + acid)
Terms modified by Tricarboxylic Acid Selected AbstractsPseudomonas fluorescens orchestrates a fine metabolic-balancing act to counter aluminium toxicityENVIRONMENTAL MICROBIOLOGY, Issue 6 2010Joseph Lemire Summary Aluminium (Al), an environmental toxin, is known to disrupt cellular functions by perturbing iron (Fe) homeostasis. However, Fe is essential for such metabolic processes as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the two pivotal networks that mediate ATP production during aerobiosis. To counter the Fe conundrum induced by Al toxicity, Pseudomonas fluorescens utilizes isocitrate lyase and isocitrate dehydrogenase-NADP dependent to metabolize citrate when confronted with an ineffective aconitase provoked by Al stress. By invoking fumarase C, a hydratase devoid of Fe, this microbe is able to generate essential metabolites. To compensate for the severely diminished enzymes like Complex I, Complex II and Complex IV, the upregulation of a H2O-generating NADH oxidase enables the metabolism of citrate, the sole carbon source via a modified TCA cycle. The overexpression of succinyl-CoA synthetase affords an effective route to ATP production by substrate-level phosphorylation in the absence of O2. This fine metabolic balance enables P. fluorescens to survive the dearth of bioavailable Fe triggered by an Al environment, a feature that may have potential applications in bioremediation technologies. [source] Isocitrate dehydrogenase of Plasmodium falciparumFEBS JOURNAL, Issue 8 2003Energy metabolism or redox control? Erythrocytic stages of the malaria parasite Plasmodium falciparum rely on glycolysis for their energy supply and it is unclear whether they obtain energy via mitochondrial respiration albeit enzymes of the tricarboxylic acid (TCA) cycle appear to be expressed in these parasite stages. Isocitrate dehydrogenase (ICDH) is either an integral part of the mitochondrial TCA cycle or is involved in providing NADPH for reductive reactions in the cell. The gene encoding P. falciparum ICDH was cloned and analysis of the deduced amino-acid sequence revealed that it possesses a putative mitochondrial targeting sequence. The protein is very similar to NADP+ -dependent mitochondrial counterparts of higher eukaryotes but not Escherichia coli. Expression of full-length ICDH generated recombinant protein exclusively expressed in inclusion bodies but the removal of 27 N-terminal amino acids yielded appreciable amounts of soluble ICDH consistent with the prediction that these residues confer targeting of the native protein to the parasites' mitochondrion. Recombinant ICDH forms homodimers of 90 kDa and its activity is dependent on the bivalent metal ions Mg2+ or Mn2+ with apparent Km values of 13 µm and 22 µm, respectively. Plasmodium ICDH requires NADP+ as cofactor and no activity with NAD+ was detectable; the for NADP+ was found to be 90 µm and that of d -isocitrate was determined to be 40 µm. Incubation of P. falciparum under exogenous oxidative stress resulted in an up-regulation of ICDH mRNA and protein levels indicating that the enzyme is involved in mitochondrial redox control rather than energy metabolism of the parasites. [source] Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyaseFEBS JOURNAL, Issue 14 2002Tadayoshi Kanao The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl -ACL), an ,4,4 heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent Km value of 0.21 ± 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (nH) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 ± 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a Ki value of 0.037 ± 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl -ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl -ACL. His273 of the , subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA. [source] Organic acids: old metabolites, new themesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2006Israel Goldberg Abstract Fumaric, L -malic and citric acids are intermediates of the oxidative tricarboxylic acid (TCA) cycle which in eukaryotes is localized in mitochondria. These organic acids are synthesized and accumulated in the medium to very high concentrations by filamentous fungi such as Aspergillus spp. and Rhizopus sp. This article reviews basic research on the unusual acid production capability and the associated metabolic pathways operating under defined stress conditions in these specific fungi. In particular, we describe and discuss the importance of the cytosolic reductive TCA pathway, which includes the cytosolic activities of pyruvate carboxylase, malate dehydrogenase and fumarase, for production of fumaric and L -malic acids. This article also describes the differences between fumaric acid, L -malic acid and citric acid production by different organisms (filamentous fungi, yeast, and higher eukaryotes), and the possible application of novel technologies (genetic engineering and bioinformatics) to fungal systems which may offer new industrial potential of filamentous fungi for the production of valuable metabolites. Copyright © 2006 Society of Chemical Industry [source] Genome-wide screen identifies Escherichia coli TCA-cycle-related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia-inducible transcription factor ArcAAGING CELL, Issue 5 2010Stavros Gonidakis Summary Single-gene mutants with extended lifespan have been described in several model organisms. We performed a genome-wide screen for long-lived mutants in Escherichia coli, which revealed strains lacking tricarboxylic acid (TCA)-cycle-related genes that exhibit longer stationary-phase survival and increased resistance to heat stress compared to wild-type. Extended lifespan in the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with the reduced production of superoxide and increased stress resistance. On the other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is associated with reduced oxygen consumption and requires the acetate-producing enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription factor ArcA, acting independently of acetate metabolism, is also required for maximum lifespan extension in the lipA and lpdA mutants, indicating that these mutations promote entry into a mode normally associated with a low-oxygen environment. Because analogous changes from respiration to fermentation have been observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans strains, such metabolic alterations may represent an evolutionarily conserved strategy to extend lifespan. [source] Brain metabolism of exogenous pyruvateJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Susana Villa Gonzalez Abstract Pyruvate given in large doses may be neuroprotective in stroke, but it is not known to what degree the brain metabolizes pyruvate. Intravenous injection of [3- 13C]pyruvate led to dose-dependent labelling of cerebral metabolites so that at 5 min after injection of 18 mmoles [3- 13C]pyruvate/kg (2 g sodium pyruvate/kg), approximately 20% of brain glutamate and GABA were labelled, as could be detected by 13C nuclear magnetic resonance spectrometry ex vivo. Pyruvate, 9 mmoles/kg, was equivalent to glucose, 9 mmoles/kg, as a substrate for cerebral tricarboxylic acid (TCA) cycle activity. Inhibition of the glial TCA cycle with fluoroacetate did not affect formation of [4- 13C]glutamate or [2- 13C]GABA from [3- 13C]pyruvate, but reduced formation of [4- 13C]glutamine by 50%, indicating predominantly neuronal metabolism of exogenous pyruvate. Extensive formation of [3- 13C]lactate from [2- 13C]pyruvate demonstrated reversible carboxylation of pyruvate to malate and equilibration with fumarate, presumably in neurones, but anaplerotic formation of TCA cycle intermediates from exogenous pyruvate could not be detected. Too rapid injection of large amounts of pyruvate led to seizure activity, respiratory arrest and death. We conclude that exogenous pyruvate is an excellent energy substrate for neurones in vivo, but that care must be taken to avoid the seizure-inducing effect of pyruvate given in large doses. [source] Advantages of deuterium-labelled mixed triacylglycerol in studies of intraluminal fat digestionRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2006Christine Slater The 13C-mixed triacylglcerol (MTG, 1,3-distearyl, 2-[1- 13C]octanoyl glycerol) breath test is a non-invasive measure of intraluminal fat digestion. Recovery of 13C in breath CO2 is incomplete (<50%) owing to sequestration of 13C into organic molecules via the tricarboxylic acid (TCA) cycle. In addition lack of knowledge of CO2 production rate (VCO2) during the test leads to errors in the calculated percentage dose recovered (PDR). 2H sequestration into organic molecules is low (,4%) and is not influenced by factors that affect VCO2 such as food intake or physical activity. After oxidation of 2H-labelled macromolecules, the label appears in body water, which can be sampled non-invasively in urine or saliva. After an overnight fast, two healthy adults consumed [2H]MTG (1,3-distearyl, 2-[2H15]octanoyl glycerol) and [13C]MTG (1,3 distearyl, 2-[1- 13C]octanoyl glycerol) simultaneously. Total body water (TBW) was measured by 18O dilution and also estimated from height and weight. Urine and saliva were sampled at baseline and for 10,h after consumption of the test meal. The abundance of 2HOH and H218O in urine and saliva was measured by continuous-flow isotope-ratio mass spectrometry. Cumulative PDR of 2H and 18O was calculated from the plateau enrichment, which was reached by 6,h in both saliva and urine. Recovery of 2H calculated using measured TBW was compared with that using an estimated value of TBW. Mean recovery of 2H in saliva was 99.3% and in urine was 96.4%. Errors introduced by estimating TBW were <5%. [2H]MTG could provide a simpler, more robust, indirect test of intraluminal fat digestion compared with the 13C-breath test. Further studies are required in pancreatic insufficient patients. Copyright © 2005 John Wiley & Sons, Ltd. [source] Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrationsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Antonino Baez Abstract The effect of dissolved carbon dioxide (dCO2) concentration on the stoichiometric and kinetic constants and by-product accumulation was determined for Escherichia coli cells producing recombinant green fluorescent protein (GFP). Constant dCO2, in the range of 20,300,mbar, was maintained during batch cultures by manipulating the inlet gas composition. As dCO2 increased, specific growth rate (µ) decreased, and acetate accumulation and the time for onset of GFP production increased. Maximum biomass yield on glucose and GFP concentration were affected for dCO2 above 70 and 150,mbar, respectively. Expression analysis of 16 representative genes showed that E. coli can respond at the transcriptional level upon exposure to increasing dCO2, and revealed possible mechanisms responsible for the detrimental effects of high dCO2. Genes studied included those involved in decarboxylation reactions (aceF, icdA, lpdA, sucA, sucB), genes from pathways of production and consumption of acetate (ackA, poxB, acs, aceA, fadR), genes from gluconeogenic and anaplerotic metabolism (pckA, ppc), genes from the acid resistance (AR) systems (adiA, gadA, gadC), and the heterologous gene (gfp). The transcription levels of tricarboxylic acid (TCA) cycle genes (icdA, sucA, sucB) and glyoxylate shunt (aceA) decreased as dCO2 increased, whereas fadR (that codes for a negative regulator of the glyoxylate operon) and poxB (that codes for PoxB which is involved in acetate production from pyruvate) were up-regulated as dCO2 increased up to 150,mbar. Furthermore, transcription levels of genes from the AR systems increased as dCO2 increased up to 150,mbar, indicating that elevated dCO2 triggers an acid stress response in E. coli cells. Altogether, such results suggest that the increased acetate accumulation and reduction in µ, biomass yield and maximum GFP concentration under high dCO2 resulted from a lower carbon flux to TCA cycle, the concomitant accumulation of acetyl-CoA or pyruvate, and the acidification of the cytoplasm. Biotechnol. Bioeng. 2009; 104: 102,110 © 2009 Wiley Periodicals, Inc. [source] A single nutrient feed supports both chemically defined NS0 and CHO fed-batch processes: Improved productivity and lactate metabolismBIOTECHNOLOGY PROGRESS, Issue 5 2009Ningning Ma Abstract A chemically defined nutrient feed (CDF) coupled with basal medium preloading was developed to replace a hydrolysate-containing feed (HCF) for a fed-batch NS0 process. The CDF not only enabled a completely chemically defined process but also increased recombinant monoclonal antibody titer by 115%. Subsequent tests of CDF in a CHO process indicated that it could also replace the hydrolysate-containing nutrient feed in this expression system as well as providing an 80% increase in product titer. In both CDF NS0 and CHO processes, the peak lactate concentrations were lower and, more interestingly, lactate metabolism shifted markedly from net production to net consumption when cells transitioned from exponential to stationary growth phase. Subsequent investigations of the lactate metabolic shift in the CHO CDF process were carried out to identify the cause(s) of the metabolic shift. These investigations revealed several metabolic features of the CHO cell line that we studied. First, glucose consumption and lactate consumption are strictly complementary to each other. The combined cell specific glucose and lactate consumption rate was a constant across exponential and stationary growth phases. Second, Lactate dehydrogenase (LDH) activity fluctuated during the fed-batch process. LDH activity was at the lowest when lactate concentration started to decrease. Third, a steep cross plasma membrane glucose gradient exists. Intracellular glucose concentration was more than two orders of magnitude lower than that in the medium. Fourth, a large quantity of citrate was diverted out of mitochondria to the medium, suggesting a partially truncated tricarboxylic acid (TCA) cycle in CHO cells. Finally, other intermediates in or linked to the glycolytic pathway and the TCA cycle, which include alanine, citrate, isocitrate, and succinate, demonstrated a metabolic shift similar to that of lactate. Interestingly, all these metabolites are either in or linked to the pathway downstream of pyruvate, but upstream of fumarate in glucose metabolism. Although the specific mechanisms for the metabolic shift of lactate and other metabolites remain to be elucidated, the increased understanding of the metabolism of CHO cultures could lead to future improvements in medium and process development. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | |||||||||||||