Showing papers on "Oxoglutarate dehydrogenase complex published in 2009"

Establishment of oxidative D-xylose metabolism in Pseudomonas putida S12

Abstract: The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose-1) and a maximum growth rate of 0.21 h -1. Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-D-xylonate dehydratase and α-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose. Copyright © 2009, American Society for Microbiology. All Rights Reserved. Chemicals / CAS: glucose dehydrogenase, 37250-49-0, 37250-50-3, 37250-84-3, 9028-53-9; hydrolyase, 9044-86-4; oxoglutarate dehydrogenase, 9031-02-1; xylose, 25990-60-7, 58-86-6; aldehyde dehydrogenase, 37353-37-0, 9028-86-8; 2,5-dioxovalerate dehydrogenase, 1.2.1.26; Aldehyde Oxidoreductases, 1.2.-; Bacterial Proteins; D-xylo-aldonate dehydratase, 4.2.1.-; Glucose 1-Dehydrogenase, 1.1.1.47; Hydro-Lyases, 4.2.1.-; Xylose

Inhibition of the alpha-ketoglutarate dehydrogenase-mediated reactive oxygen species generation by lipoic acid

Abstract: Dihydrolipoamide dehydrogenase (LADH) is a flavo-enzyme that serves as a subunit of alpha-ketoglutarate dehydrogenase complex (alpha-KGDHC). Reactive oxygen species (ROS) generation by alpha-KGDHC has been assigned to LADH (E3 subunit) and explained by the diaphorase activity of E3. Dysfunctions of alpha-KGDHC and concurrent ROS production have been implicated in neurodegeneration, ischemia-reperfusion, and other pathological conditions. In this work we investigated the in-depth details of ROS generation by isolated LADH and alpha-KGDHC. We found a parallel generation of superoxide and hydrogen peroxide by the E3 subunit of alpha-KGDHC which could be blocked by lipoic acid (LA) acting on a site upstream of the E3 subunit. The pathologically relevant ROS generation (at high NADH/NAD+ ratio and low pH) in the reverse mode of alpha-KGDHC could also be inhibited by LA. Our results contradict the previously proposed mechanism for pH-dependent ROS generation by LADH, showing no disassembling of the E3 functional homodimer at acidic pH using a physiologically relevant method for the examination. It is also suggested that LA could be beneficial in reducing the cell damage related to excessive ROS generation under pathological conditions.

Small-molecule targeting of the mitochondrial compartment with an endogenously cleaved reversible tag.

Abstract: Targeted accumulation of chemically unaltered compounds within the mitochondrial compartment has not yet been achieved. Here we describe a reversible tag that is endogenously cleaved after mitochondrial accumulation has occurred. Specifically, we have reversibly tagged alpha-lipoic acid with a triphenylphosphonium moiety that is cleaved by the physiologically contained mitochondrial aldehyde dehydrogenase (ALDH-2). This reversibly tagged compound activates the lipoic acid-sensitive pyruvate dehydrogenase complex, and this results in increased glucose oxidation. We observed a reduction in ROS accumulation after preincubation with the reversibly tagged compound, whereas untagged or irreversibly tagged compounds either had no effect on ROS formation or rather caused increased oxidative stress, respectively. Lastly, the cytotoxicity of the reversibly tagged compound is less than that of the irreversibly tagged compound. Overall, reversible tagging combines decreased tag-related cytotoxicity with increased bioactivity, and this potentially provides a novel concept in mitochondrial pharmacology.

Defects in citric acid cycle and the electron transport chain in progressive poliodystrophy.

Abstract: We will present 8 children with progressive infantile or juvenile poliodystrophy (Alpers' disease), associated with a defect in pyruvate metabolism. Laboratory studies showed elevated levels of lactate in CSF and, in 4 children, elevated levels in serum. Histopathologic studies revealed lipid storage in liver and/or muscle tissue, sometimes myopathy with abnormal mitochondria and slight axonal degeneration in the peripheral nerve. Autopsy showed the characteristics of progressive poliodystrophy with degeneration and loss of neurons. Electron microscopy of cerebral cortex showed no mitochondrial abnormalities in neurons or astroglia. Biochemical studies in muscle and/or liver and/or cerebral tissue showed different deficiencies in pyruvate metabolism: in the pyruvate dehydrogenase complex, in the second part of the citric acid cycle (after the oxoglutarate dehydrogenase complex), in the NADH oxidation, in cytochrome aa3 and in pyruvate carboxylase.

Ca2+ -independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca2+ and Mg2+.

Abstract: In the absence of exogenous Ca2+ and Mg2+ and in the presence of EGTA, which favours the release of endogenous Ca2+, the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E1α subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E1α phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP2 and at high concentrations it stimulates PDK2.

Physiological role and regulation of glutamate dehydrogenase in Prochlorococcus sp. strain MIT9313

Abstract: Summary Glutamate dehydrogenase is an enzyme catalysing a reaction for ammonium assimilation, alternative to those performed by glutamine synthetase and glutamate synthase. In the genus Prochlorococcus, genomic studies have shown the presence of the gdhA gene (encoding glutamate dehydrogenase) in only four of the sequenced strains, including MIT9313. We studied the physiological regulation of glutamate dehydrogenase in this strain, by measuring the expression of gdhA, the intracellular concentration of the enzyme and its activity. Our goal was to clarify the physiological role of glutamate dehydrogenase, in order to understand why it has been selectively conserved in certain strains. Studies performed in cultures under nitrogen starvation, or with inhibitors of the nitrogen assimilation, suggest that the main role of glutamate dehydrogenase is not the assimilation of ammonium. Glutamate dehydrogenase activity and gdhA expression increased along the growth of cultures. Besides, we found a significant upregulation in gene expression when cultures were grown on glutamate as nitrogen source. We suggest that the main physiological role of glutamate dehydrogenase in Prochlorococcus MIT9313 is the utilization of glutamate to produce ammonium and 2-oxoglutarate, and amino acid recycling, thus enabling to use amino acids as nitrogen source. Therefore we propose that glutamate dehydrogenase is present in the genome of strains for whom the utilization of amino acids is most important.

Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-Phosphorylation of Pyruvate Dehydrogenase:

Abstract: A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H2O2, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.

Mutant formate dehydrogenase, gene encoding same, and method for producing nadh

Abstract: Durability of formate dehydrogenase is improved with the use of formate dehydrogenase exhibiting high specific activity that is unpredictable from conventional findings. A specific amino acid substitution is introduced into Gibberella zeae -derived formate dehydrogenase. Mutant formate dehydrogenase exhibits durability that is extremely superior to that of wild-type formate dehydrogenase. Thus, the productivity of NADH that is produced using the mutant formate dehydrogenase can be improved.

Significant reduction of erythrocyte glucose-6-phosphate dehydrogenase activity in soccer-players during play. Evidence for catecholamine mediated enzyme inhibition.

Abstract: We previously reported that mild reductions in erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity normally induced by training of basketball players was prevented by the administration of the antioxidants L-cysteine or a-tocopherol (1, 2). Because high catecholamine concentrations are reported to contribute to total antioxidant status (TAS) (3), and since G6PD activity is closely related to oxidative stress, we investigated enzyme activity in soccer players at the intermission and post-game in relation to their TAS and blood catecholamine concentrations. This study was approved by the Greek Ethical Committee as amended in 1989. Fourteen, 20-year-old soccer players who were actively participating in the Greek Soccer Championship volunteered to participate. They had trained for at least two years, consisting of two to three 60-min sessions per week, for nine months each year. A championship game took place on a 60 m=40 m pitch. Each team was comprised of seven players; two forwards, two mildfielders, two defenders and one goalkeeper. To ensure similar technical ability between each team, opponents were chosen with a similar league position to the ‘‘experimental’’ team. Clinical and biochemical blood parameters were assessed pre-game (3–4 min prior to start), at the beginning of half-time and at the end of the game (within 3–4 min post-game). Capillary blood samples were obtained from the left thumb for ana-

Characterization of the interaction between rat pyruvate dehydrogenase kinase 4 and adp

Abstract: .......................................... ............................................... iii Acknowledgements................................... ...................................... v Table of

Regulatory Role of Supramolecular Alcohol Dehydrogenase and Lactate Dehydrogenase Complex in Cell Mitochondria

Abstract: The existence of a supramolecular alcohol dehydrogenase–lactate dehydrogenase complex was demonstrated. Enzyme activities were evaluated in a preparation of mitochondrial membranes from mouse liver. The role of the enzyme complex (alcohol dehydrogenase–lactate dehydrogenase) in the regulation of pyruvate and acetaldehyde metabolism in the cell was studied.

The catabolism of glucose, glutamate, pyruvate and acetate in neisseria elongata subsp. glycolytica

Abstract: Activities corresponding to the enzymes glucokinase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, malate dehydrogenase, pyridine nucleotide independent malate dehydrogenase, and glutamate dehydrogenase were found in cell free extracts from Neisseria elongata subsp. gkcolytica. Activities corresponding to 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase were not found. Glucose was catabolized only vira the pentose phosphate pathway. The radiorespirometric findings suggest an extensive recycling of the triose and fructose phosphates. There was no evidence for formation of pyruvate from glucose. Glutamate was oxidized via the tricarboxylic acid cycle. Pyruvate and acetate were obviously catabolized by the glyoxylic and tricarboxylic acid cycles, as in N. elongata.