A Comparative Study of the Isocitrate Dehydrogenases of Chlorobium limicola forma thiosulfatophilum and Rhodopseudomonas palustris

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in enzymatic and kinetic properties.     

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli

Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.     

Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle.

Isocitrate dehydrogenase (IDH) catalyzes the reversible conversion between isocitrate and 2-oxoglutarate accompanied by decarboxylation/carboxylation and oxidoreduction of NAD(P)+ cofactor. While this enzyme has been well studied as a catabolic enzyme in the tricarboxylic acid (TCA) cycle, here we have characterized NADP-dependent IDH from Chlorobium limicola, a green sulfur bacterium that fixes CO2 through the reductive tricarboxylic acid (RTCA) cycle, focusing on the CO2-fixation ability of the enzyme. The gene encoding Cl-IDH consisted of 2226 bp, corresponding to a polypeptide of 742 amino acid residues. The primary structure and the size of the recombinant protein indicated that Cl-IDH was a monomeric enzyme of 80 kDa distinct from the dimeric NADP-dependent IDHs predominantly found in bacteria or eukaryotic mitochondria. Apparent Michaelis constants for isocitrate (45 +/- 13 microm) and NADP+ (27 +/- 10 microm) were much smaller than those for 2-oxoglutarate (1.1 +/- 0.5 mm) and CO2 (1.3 +/- 0.3 mm). No significant differences in kinetic properties were observed between Cl-IDH and the dimeric, NADP-dependent IDH from Saccharomyces cerevisiae (Sc-IDH) at the optimum pH of each enzyme. However, in contrast to the 20% activity of Sc-IDH toward carboxylation as compared with that toward decarboxylation at pH 7.0, the activities of Cl-IDH for both directions were almost equivalent at this pH, suggesting a more favorable property of Cl-IDH than Sc-IDH as a CO2-fixation enzyme under physiological pH. Furthermore, we found that among various intermediates, oxaloacetate was a competitive inhibitor (K(i) = 0.35 +/- 0.04 mm) for 2-oxoglutarate in the carboxylation reaction by Cl-IDH, a feature not found in Sc-IDH.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.     

ENZYMES OF THE TRICARBOXYLIC ACID CYCLE IN SEA URCHIN EGGS AND EMBRYOS

Changes in the activity of some enzymes of the tricarboxylic acid cycle during development of sea urchins were investigated. Unfertilized eggs showed substantial activity of citrate synthase, aconitase, NAD- and NADP-specific isocitrate dehydrogenases, fumarase and malate dehydrogenase. During development, the activity of citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase and malate dehydrogenase increases gradually, whereas the activity of fumarase remains rather constant. There is no close correlation between changes in the enzyme activity and the increase in oxygen consumption during development. Citrate synthase, aconitase, NADP-specific isocitrate dehydrogenase are mainly localized in the mitochondrial fraction, whereas fumarase and malate dehydrogenase are present in both mitochondrial and cytosol fractions. The intracellular localization of these enzymes does not change during development. A possible mechanism for the regulation of some enzymes of the tricarboxylic acid cycle in sea urchin eggs is discussed.     

Effects of metabolic substrates and inhibitors on weak organic anion transport in rat renal proximal tubules

Stimulatory effects of intermediates of the tricarboxylic acid cycle on renal uptake of a weak organic anion, fluorescein, were studied with the aid of the method of contact microfluorimetry of individual convoluted proximal tubules ascending to the surface of the rat renal cortex slices. The study was undertaken for verifying the hypothesis that energization of renal excretion of anionic exenobiotics is mediated through their transport across the basolateral membrane in exchange for cytoplasmic alpha-ketoglutarate serving as a counter-anion. Effects of inhibitors of the tricarboxylic acid cycle such as fluoroacetate, malonate and 5-methoxyindole-2-carboxylate on the fluorescein uptake and renal gluconeogenesis in the presence of the metabolic substrates were investigated in order to outline metabolic pathways that could be responsible for elevation of the cytoplasmic alpha-ketoglutarate. Obtained data evidence that the stimulatory effects of the tricarboxylic acid cycle intermediates on the transport process under study depend on the metabolic state of the mitochondria and involve an activation of certain reactions but not the cycle as a whole. It has been suggested that an elevation of the cytoplasmic alpha-ketoglutarate resulting from this activation can be conditioned by export of isocitrate from the mitochondria with its subsequent transformation into alpha-ketoglutarate in the cytoplasm in the isocitrate dehydrogenase reaction.     

Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development

The specific activities of the enzymes of the tricarboxylic acid cycle; citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were determined in early fifth-stage, young and mature adult Obeliscoides cuniculi, the rabbit stomach worm. ∝-Ketoglutarate dehydrogenase activity could not be determined in any fraction. Fumarate reductase activity was found only in the mitochondrial fraction while all other enzymes, including an NADP-dependent malic enzyme were localized in the cytoplasm. Glutamate dehydrogenase, acid and alkaline phosphatase activities were also recorded. High levels of those enzymes acting in the “reversed” direction, i.e. MDH and fumarase relative to the enzymes of the “forward” direction, i.e. citrate synthase, aconitase and isocitrate dehydrogenase suggests that under anaerobic conditions a modified tricarboxylic acid cycle can operate. Some variations in specific activities were apparent as the worms matured but no qualitative differences were observed.     

Carbohydrate catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The plerocercoids of S. solidus possess a complete sequence of glycolytic and tricarboxylic acid cycle enzymes. The presence of phosphoenolpyruvate carboxykinase and fumarate reductase activity and the relatively low activities of aconitase and isocitrate dehydrogenase suggest that carbon dioxide fixation is an important pathway in this parasite. Carbon balances show that glycogen is the main energy source under both aerobic and anaerobic conditions and there is only a slight Pasteur effect. Aerobically 22·5% of the glycogen catabolized is excreted as acetate and propionate (4:1), anaerobically 70% of the glycogen utilized can be accounted for as acetate and propionate (1:3). The results indicate that anaerobically the plerocercoids fix carbon dioxide and have a partial reversed tricarboxylic acid cycle, whilst under aerobic conditions at least part of the carbohydrate may be oxidized via a functional tricarboxylic acid cycle.     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

Subunit structure, expression, and function of NAD(H)-specific isocitrate dehydrogenase in Saccharomyces cerevisiae.

Mitochondrial NAD(H)-specific isocitrate dehydrogenase was purified from Saccharomyces cerevisiae for analyses of subunit structure and expression. Two subunits of the enzyme with different molecular weights (39,000 and 40,000) and slightly different isoelectric points were resolved by denaturing electrophoretic techniques. Sequence analysis of the purified subunits showed that the polypeptides have different amino termini. By using an antiserum to the native enzyme prepared in rabbits, subunit-specific immunoglobulin G fractions were obtained by affinity purification, indicating that the subunits are also immunochemically distinct. The levels of NAD(H)-specific isocitrate dehydrogenase activity and immunoreactivity were found to correlate closely with those of a second tricarboxylic acid cycle enzyme, malate dehydrogenase, in yeast cells grown under a variety of conditions. S. cerevisiae mutants with defects in NAD(H)-specific isocitrate dehydrogenase were identified by screening a collection of yeast mutants with acetate-negative growth phenotypes. Immunochemical assays were used to demonstrate that one mutant strain lacks the 40,000-molecular-weight subunit (IDH1) and that a second strain lacks the 39,000-molecular-weight subunit (IDH2). Mitochondria isolated from the IDH1 and IDH2 mutants exhibited a markedly reduced capacity for utilization of either isocitrate or citrate for respiratory O2 consumption. This confirms an essential role for NAD(H)-specific isocitrate dehydrogenase in oxidative functions in the tricarboxylic acid cycle.     

Pyruvate and citrate metabolism in the muscle tissue of Ascaris lumbricoides.

Aconitase and NAD linked isocitrate dehydrogenase were present in Ascaris lumbricoides muscle at only very low activities, whilst there were significant levels of citrate synthase, NADP linked isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase and succinic thiokinase. Pyruvate dehydrogenase was present in A. lumbricoides muscle at levels comparable with mammalian tissues and results suggest that it is modulated via a phosphotransferase/phosphatase system. The tricarboxylic acid cycle intermediates, citrate, isocitrate and 2-oxoglutarate were all detected in freeze clamped muscle, but their steady state levels were considerably lower than those found in mammalian tissues.     

The acid end-products of glucose metabolism of oral and other haemophili

The acids produced in broth culture by various species of oral haemophili and by stock strains of capsulated and other haemophili were identified and measured by gas-liquid chromatography. Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly produced but in smaller amounts. A stock strain, Haemophilus parainfluenzae NCTC 4101, produced less succinic acid than other strains of haemophili. Strain NCTC 4101 possessed all the enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected. No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase activity appear to be characteristic of haemophili. The pathway to succinate in haemophili appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate and fumarate. The results of tracer studies on a single oral strain of H. parainfluenzae using various labelled substrates were in keeping with this proposed metabolic pathway.     

Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens.

Oxalic acid plays a pivotal role in the adaptation of the soil microbe Pseudomonas fluorescens to aluminum (Al) stress. Its production via the oxidation of glyoxylate necessitates a major reconfiguration of the enzymatic reactions involved in the tricarboxylic acid (TCA) cycle. The demand for glyoxylate, the precursor of oxalic acid appears to enhance the activity of isocitrate lyase (ICL). The activity of ICL, an enzyme that participates in the cleavage of isocitrate to glyoxylate and succinate incurred a 4-fold increase in the Al-stressed cells. However, the activity of isocitrate dehydrogenase, a competitor for the substrate isocitrate, appeared to be diminished in cells exposed to Al compared to the control cells. While the demand for oxalate in Al-stressed cells also negatively influenced the activity of the enzyme alpha-ketoglutarate dehydrogenase complex, no apparent change in the activity of malate synthase was recorded. Thus, it appears that the TCA cycle is tailored in order to generate the necessary precursor for oxalate synthesis as a consequence of Al-stress.     

Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin-degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin

A proteomic differential display technique was utilized to study cellular responses of Phanerochaete chrysosporium exposed to vanillin, one of the key intermediates found during lignin biodegradation. Intracellular proteins were resolved by 2-DE and target protein spots were identified using MALDI-MS after in-gel tryptic digestions. Upon addition of vanillin to P. chrysosporium, up-regulation of homogentisate 1,2-dioxygenase, 1,4-benzoquinone reductases, aldehyde dehydrogenase, and aryl-alcohol dehydrogenase, which seem to play roles in vanillin metabolism, was observed. Furthermore, enzymes involved in glycolysis, the tricarboxylic acid cycle, the pentose-phosphate cycle, and heme biosynthesis were also activated. Up-regulation of extracellular peroxidase was also observed. One of the most unique phenomena against exogenous vanillin was a switch from the glyoxylate cycle to the tricarboxylic acid cycle, where a drastic increase in isocitrate dehydrogenase activity was observed. The exogenous addition of other aromatic compounds also caused an increase in its activity, which in turn triggered NAD(P)H production via the action of dehydrogenases in the tricarboxylic acid cycle, heme biosynthesis via the action of aminolevulinic acid synthase on succinyl-CoA, and energy production via activation of the mitochondrial electron transfer system. These metabolic shifts seem to be required for activating a metabolic system for aromatic compounds.     

Phosphoenolpyruvate-succinate-glyoxylate pathway in the filarial parasiteSetaria digitata

Setaria digitata, a filarial parasite of cattle possesses certain unique characteristics like cyanide insensitivity, and lack of cytochromes. In the present study, we have shown that the parasite has an incomplete tricarboxylic acid cycle with the absence of activities of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthase. However the parasite showed the existence of glyoxylate cycle and phosphoenolpyruvate-succinate pathway. The widely used antifilarial drug diethylcarbamazine caused general inhibition of all enzymes of phosphoenolpyruvate-succinate pathway and glyoxylate cycle except that of fumarase and isocitrate lyase. The results may pave the way for new targets for chemotherapy in the control of filarial parasites.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

Chemostat culture of Bacillus caldotenax at 65°C: Activity and regulation of the main metabolic pathways

Bacillus caldotenax was cultivated in chemostat experiments at 65°C with a chemically defined minimal medium. Glycolysis, tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain were active as demonstrated by measuring the corresponding enzymes. No enzyme activity of the Entner-Doudoroff pathway could be detected. The specific activities of the citrate cycle enzymes were up to 10 times higher as compared to the enzymes of glycolysis. At dilution rates between 0.3 and 2.2 h^-1 none of the main metabolic pathways was regulated. In contrast the isocitrate lyase was regulated (drop of activity with increasing growth rates). As a result of a batch culture with glucose and acetate as carbon sources a regulation model was proposed: glucose, or a metabolite of glucose, represses the isocitrate lyase; in the absence of glucose acetate acts as an inducer.Abbreviations DCIP dichlorphenol indophenol - ED Entner-Doudoroff pathway - EMP Emden-Meyerhof-Parnas pathway - ICL isocitrate lyase - PP pentose phosphate pathway - TCC tricarbonic acid cycle     

Metabolism of Yarrowia lipolytica grown on ethanol under conditions promoting the production of alpha-ketoglutaric and citric acids: a comparative study of the central metabolism enzymes

A comparative study of the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles in the mutant Yarrowia lipolytica strain N1 capable of producing alpha-ketoglutaric acid (KGA) and citric acid showed that almost all enzymes of the TCA cycle are more active under conditions promoting the production of KGA. The only exception was citrate synthase, whose activity was higher in yeast cells producing citric acid. The production of both acids was accompanied by suppression of the glyoxylate cycle enzymes. The activities of malate dehydrogenase, aconitase, NADP-dependent isocitrate dehydrogenase, and fumarase were higher in cells producing KGA than in cells producing citric acid.     

Purification and characterization of NADP-linked isocitrate dehydrogenase from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris

NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the Km of 12.7, 2.9, and 23.9 microM for isocitrate, NADP, and Mg2+, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg2+, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (K(i), 127.0 microM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.