2-oxoglutarate Dehydrogenase Activity Research Articles

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Ferrous sulfate reverses cerebral metabolic abnormality induced by minimal hepatic encephalopathy.

Orally administered ferrous iron was previously reported to significantly improve the cognition and locomotion of patients with minimal hepatic encephalopathy (MHE). However, the metabolic mechanisms of the therapeutic effect of ferrous iron are unknown. In this study, MHE was induced in rats by partial portal vein ligation (PPVL), and was treated with ferrous sulfate. The Morris water maze was used to evaluate the cognitive condition of the rats. The metabolites observed by NMR and validated by liquid chromatography-mass spectrometry were defined as the key affected metabolites. The enzyme activities and trace element contents in the rat brains were also investigated. The Mn content was found to be increased but the ferrous iron content decreased in the cortex and striatum in MHE. Decreased oxoglutarate dehydrogenase activity and increased glutamine synthetase (GS) and pyruvate carboxylase (PC) activity were observed in the cortex of MHE rats. Decreased pyruvate dehydrogenase activity and increased GS and PC activity were observed in the striatum of MHE rats. The levels of BCAAs and taurine were significantly decreased, and the contents of GABA, lactate, arginine, aspartate, carnosine, citrulline, cysteine, glutamate, glutamine, glycine, methionine, ornithine, proline, threonine and tyrosine were significantly increased. These metabolic abnormalities described above were restored after treatment with ferrous sulfate. Pathway enrichment analysis suggested that urea cycle, aspartate metabolism, arginine and proline metabolism, glycine and serine metabolism, and glutamate metabolism were the major metabolic abnormalities in MHE rats, but these processes could be restored and cognitive impairment could be improved by ferrous sulfate administration.

Arbuscular mycorrhiza fungus alleviates arsenic mediated disturbances in tricarboxylic acid cycle and nitrogen metabolism in Triticum aestivum L.

Utilization of arbuscular mycorrhizal (AM) fungi (AMF) as a sustainable strategy in redeeming arsenic (As) toxicity in plants is a promising approach. Low As accumulation, restoration of physiological processes, and As tolerance by AMF have been documented in crop plants. However, to comprehend AM-mediated As tolerance in plants, understanding the biochemical responses of host to the symbiont is crucial. The study evaluated the effect of an AM fungus, Rhizophagus intraradices on tricarboxylic acid cycle (TCA) and nitrogen metabolism of Triticum aestivum under three As concentrations (0, 25, and 50 mg As kg−1 soil). Results showed that TCA cycle and nitrogen metabolism were severely impaired by As that resulted into a higher C/N ratio. However, colonization by R. intraradices attenuated As mediated alterations in TCA cycle by augmenting the activity of pyruvate dehydrogenase that provided sufficient substrate for the TCA cycle. Furthermore, mycorrhizal (M) plants reinstated the activities of isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase even under high As level. Although citrate synthase and oxoglutarate dehydrogenase activities declined upon As exposure in M-plants, these were nevertheless higher than their non-mycorrhizal (NM) counterparts, ensuring higher levels of citric acid and succinic acid in M-plants. AM colonization also moderated the As-mediated disturbances in nitrogen assimilation by augmenting the activity of nitrate reductase, nitrite reductase, glutamine synthase, and glutamine-2-oxoglutarate amino transferase. Overall findings of the study point out that colonization by R. intraradices favourably regulated the TCA cycle and nitrogen metabolism and confronted As-mediated alterations in C/N ratio.

A combination of simvastatin and low-protein diet increases renal 2-oxoglutarate dehydrogenase activity in rats

Mitochondrial 2-oxoglutarate dehydrogenase complex (2-OGDH) that consists of multiple copies of 3 catalytic subunits (E1, E2, E3) is a regulatory enzyme of tricarboxylic acid cycle. 2-OGDH together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) belongs to the 2-oxoacid dehydrogenases family. It was shown that in protein-restricted rats simvastatin stimulated liver BCKDH, whereas it exerted no effect on BCKDH in rats fed a standard diet. We hypothesized that combination of simvastatin and low-protein diet could have an impact on renal 2-OGDH. The purpose of the study was to determine the effect of combination of simvastatin and low-protein diet on renal 2-OGDH in rats. Simvastatin (80 mg/kg b.wt/day) or the vehicle (0.3% methylcellulose) were administered orally (for 14 days) to rats fed low-protein (8% protein) or standard (23% protein) diet. 2-OGDH activity, protein levels and mRNA levels for E1 and E2 subunits were determined. In addition, serum creatinine level was measured. Results: The combination of simvastatin and low-protein diet elicited the increase of renal 2-OGDH activity that corresponded to the increase of E1 protein, but not of E1 mRNA level. In contrast, simvastatin treatment did not affect 2-OGDH activity, nor protein and mRNA levels of E1 in rats fed standard diet. Serum creatinine levels were not changed upon simvastatin administration in any group. In conclusion, the results of present study indicate that combination of simvastatin and low-protein diet induces stimulation of renal 2-OGDH complex probably at post-transcriptional level.

Characteristics of the GlnH and GlnX Signal Transduction Proteins Controlling PknG-Mediated Phosphorylation of OdhI and 2-Oxoglutarate Dehydrogenase Activity in Corynebacterium glutamicum.

In Corynebacterium glutamicum the protein kinase PknG phosphorylates OdhI and thereby abolishes the inhibition of 2-oxoglutarate dehydrogenase activity by unphosphorylated OdhI. Our previous studies suggested that PknG activity is controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX, because ΔglnH and ΔglnX mutants showed a growth defect on glutamine similar to that of a ΔpknG mutant. We have now confirmed the involvement of GlnH and GlnX in the control of OdhI phosphorylation by analyzing the OdhI phosphorylation status and glutamate secretion in ΔglnH and ΔglnX mutants and by characterizing ΔglnX suppressor mutants. We provide evidence for GlnH being a lipoprotein and show by isothermal titration calorimetry that it binds l-aspartate and l-glutamate with moderate to low affinity, but not l-glutamine, l-asparagine, or 2-oxoglutarate. Based on a structural comparison with GlnH of Mycobacterium tuberculosis, two residues critical for the binding affinity were identified and verified. The predicted GlnX topology with four transmembrane segments and two periplasmic domains was confirmed by PhoA and LacZ fusions. A structural model of GlnX suggested that, with the exception of a poorly ordered N-terminal region, the entire protein is composed of α-helices and small loops or linkers, and it revealed similarities to other bacterial transmembrane receptors. Our results suggest that the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade serves to adapt the flux of 2-oxoglutarate between ammonium assimilation via glutamate dehydrogenase and energy generation via the tricarboxylic acid (TCA) cycle to the availability of the amino group donors l-glutamate and l-aspartate in the environment. IMPORTANCE Actinobacteria comprise a large number of species playing important roles in biotechnology and medicine, such as Corynebacterium glutamicum, the major industrial amino acid producer, and Mycobacterium tuberculosis, the pathogen causing tuberculosis. Many actinobacteria use a signal transduction process in which the phosphorylation status of OdhI (corynebacteria) or GarA (mycobacteria) regulates the carbon flux at the 2-oxoglutarate node. Inhibition of 2-oxoglutarate dehydrogenase by unphosphorylated OdhI shifts the flux of 2-oxoglutarate from the TCA cycle toward glutamate formation and, thus, ammonium assimilation. Phosphorylation of OdhI/GarA is catalyzed by the protein kinase PknG, whose activity was proposed to be controlled by the periplasmic binding protein GlnH and the transmembrane protein GlnX. In this study, we combined genetic, biochemical, and structural modeling approaches to characterize GlnH and GlnX of C. glutamicum and confirm their roles in the GlnH-GlnX-PknG-OdhI-OdhA signal transduction cascade. These findings are relevant also to other Actinobacteria employing a similar control process.

The Brain Protein Acylation System Responds to Seizures in the Rat Model of PTZ-Induced Epilepsy.

Abnormal energy expenditure during seizures and metabolic regulation through post-translational protein acylation suggest acylation as a therapeutic target in epilepsy. Our goal is to characterize an interplay between the brain acylation system components and their changes after seizures. In a rat model of pentylenetetrazole (PTZ)-induced epilepsy, we quantify 43 acylations in 29 cerebral cortex proteins; levels of NAD+; expression of NAD+-dependent deacylases (SIRT2, SIRT3, SIRT5); activities of the acyl-CoA-producing/NAD+-utilizing complexes of 2-oxoacid dehydrogenases. Compared to the control group, acylations of 14 sites in 11 proteins are found to differ significantly after seizures, with six of the proteins involved in glycolysis and energy metabolism. Comparing the single and chronic seizures does not reveal significant differences in the acylations, pyruvate dehydrogenase activity, SIRT2 expression or NAD+. On the contrary, expression of SIRT3, SIRT5 and activity of 2-oxoglutarate dehydrogenase (OGDH) decrease in chronic seizures vs. a single seizure. Negative correlations between the protein succinylation/glutarylation and SIRT5 expression, and positive correlations between the protein acetylation and SIRT2 expression are shown. Our findings unravel involvement of SIRT5 and OGDH in metabolic adaptation to seizures through protein acylation, consistent with the known neuroprotective role of SIRT5 and contribution of OGDH to the Glu/GABA balance perturbed in epilepsy.

FRI-1 Is an Anti-Cancer Isoquinolinequinone That Inhibits the Mitochondrial Bioenergetics and Blocks Metabolic Shifts by Redox Disruption in Breast Cancer Cells.

Since breast cancer (BC) cells are dependent on mitochondrial bioenergetics for promoting proliferation, survival, and metastasis, mitochondria highlight as an important target for anticancer drug discovery. FRI-1, methyl 1, 3-dimethyl-5, 8-dioxo-5, 8-dihydro-4-isoquinolinecarboxylate, was previously described as a selective cytotoxic compound on cancer cell lines, however, details on the mechanism of action remain unknown. In this work, we describe that FRI-1 inhibits mitochondrial bioenergetics, producing apoptosis in MCF7 and MDA-MB-231 BC cell lines. FRI-1 decreases the maximal oxygen consumption rate (OCR), Δψm, NADH, and ATP levels, with a notable increase of mitochondrial reactive oxygen species (ROS) production, promoting AMPK activation with pro-survival effects. Moreover, FRI-1 inhibits the metabolic remodeling to glycolysis induced by oligomycin. In isolated tumoral mitochondria, FRI-1 increases Complex I and III-dependent OCR state 2, and this is sensitive to rotenone and antimycin A inhibitor additions, suggesting a redox cycling event. Remarkably, α-ketoglutarate and lipoic acid supplementation reversed and promoted, respectively, the FRI-1-induced apoptosis, suggesting that mitochondrial redox disruption affects 2-oxoglutarate dehydrogenase (OGDH) activity, and this is involved in their anticancer mechanism. Consistent with this, the combination of FRI-1 and CPI-613, a dual inhibitor of redox-sensible tricarboxylic acid (TCA) cycle enzymes PDH and OGDH, produced extensive BC cell death. Taken together, our results suggest that FRI-1 exhibits anticancer effects through inhibition of mitochondrial bioenergetics by redox disruption in BC cells.

Role of Epigenetic Mechanisms in Regulating the Activity of 2-OGDH and MDH in Maize Leaves (Zea mays L.) during Hypoxia

Molecular and epigenetic ways of regulating the activity of key TCA enzymes, 2-oxoglutarate dehydrogenase (2-OGDH) and malate dehydrogenase (MDH), in maize (Zea mays L.) leaves under hypoxic conditions were studied. It has been shown that regulation of the rate of enzyme functioning under stress conditions is caused not by conformational transformations of protein molecules but by changes in the transcriptional activity of their genes. Analysis into the level of transcripts of genes encoding 2-OGDH and MDH revealed a correlation with changes in the total enzymatic activity. When plants were incubated under hypoxic conditions, a decrease in the expression of the 2-OGDH and MDH genes was observed. It was found that fluctuations in the content of gene transcripts ogdh-1 and ogdh-3 is associated with a change in the methyl status of CG-dinucleotides in their promoters. An increase in the expression of these genes is associated with a decrease in the degree of methylation of their promoters. Conversely, a decrease in the relative level of transcripts is caused by an increase in the amount of methylated CG-dinucleotides. It is concluded that regulating the functioning of 2-oxoglutarate dehydrogenase and malate dehydrogenase under conditions of low oxygen concentrations takes place by means of an epigenetic mechanism, that is, by changing the methyl status of their gene promoters.

IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis

AbstractIsocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl–coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.

Вплив вікасолу на активність дегідрогеназ циклу Кребса та стан системи антиоксидантного захисту м’язів шлунка гусей

The aim of work was to study the effect of vicasol on the Krebs cycle dehydrogenase activity and the state of the antioxidant defense system of geese muscle tissue of the stomach. According to the results of the work, it was found in the smooth muscle tissue of the stomach of geese, vicasol increases the activity of glutathione peroxidase, catalase and superoxide dismutase on the 14th and 21st days of ontogenesis, in particular, on the 14th day glutathione peroxidase, catalase and superoxide dismutase activity increase by 181.1 % (p≤0.05), 153.2% (p≤0.05) i 103.3% (p≤0.05), after 7 days 168.0% (p≤0.05), 99.7% (p≤0.05) i 111.3% (p≤0.05) relative to the control group. The antioxidant system enzymes activity of the experimental group on the 28th day tends to decrease, however, a significant difference is observed only between groups of animals for superoxide dismutase, whose activity is reduced by 62.5 % under the influence of vicasol (p≤0.05). On the 35th day of ontogenesis, the activity of glutathione peroxidase and catalase in the experimental group increases by 43.2% (p≤0.05) and 54.1% (p≤0.05) compared to the control, at this time the activity of superoxide dismutase is lower by 23.9 % (p≤0.05). A significant increase in the concentration of lipids hydrogen peroxides (28th day) by 27% (p≤0.05) relative to the control was established in the tissue, followed by a decrease on the 35th day of ontogenesis. Vicasol stabilizes the content of end products of lipid peroxidation in tissue homogenate, with the exception of a decrease of 8.6% (p≤0.05) at the end of experiment. Upon induction of peroxide processes with Fe 2+ in cell, it is increased their content by 108 % (p≤0.05) (21st day), at the end of the experiment, a decrease in the content of secondary lipid decomposition products in the experimental group of animals by 27.5% (p≤0.05). The antioxidant activity of the tissue at the beginning of the experiment under the influence of vicasol was higher by 30%, on the the 21st day decreased by 50% relative to the control group, and on the 35th day it increased by 21,6 %. Vicasol increases the activity of the Krebs cycle dehydrogenase, in particular, succinate dehydrogenase, which activity is higher during the experiment by 14- (78.2%; p≤0.05), 21- (263.3%; p≤0.05), 28- (78, 8%; p≤0.05) and the 35th (92.3%; p≤0.05) day. 2-oxoglutarate dehydrogenase activity are more specific and significantly increases relative to the control group by 14- (122.2%; p≤0.05), 21- (84.4%; p≤0.05) and on the 28th (133.3%; p≤ 0.05) day relative to the control group. In general, vicasol activates the system of antioxidant protection and energy metabolism, that is, it is a complex activator of the body’s metabolic functions.

Triheptanoin alters [U-13C6]-glucose incorporation into glycolytic intermediates and increases TCA cycling by normalizing the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in a chronic epilepsy mouse model

Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-13C6]-glucose (i.p.), enrichments of 13C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of 13C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of 13C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of 13C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or – as an additional astrocytic fuel – it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of 13C was reduced in the interictal state. Triheptanoin restored the correlation between 13C enrichments of pyruvate relative to most of the TCA cycle intermediates in “epileptic” mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

The role of endogenous thiamine produced via THIC in root nodule symbiosis in Lotus japonicus

Thiamine is a pivotal primary metabolite which is indispensable to all organisms. Although its biosynthetic pathway has been well documented, the mechanism by which thiamine influences the legume-rhizobium symbiosis remains uncertain. Here, we used overexpressing transgenic plants, mutants and grafting experiments to investigate the roles played by thiamine in Lotus japonicus nodulation. ljthic mutants displayed lethal phenotypes and the defect could be overcome by supplementation of thiamine or by overexpression of LjTHIC. Reciprocal grafting between L. japonicus wild-type Gifu B-129 and ljthic showed that the photosynthetic products of the aerial part made a major contribution to overcoming the nodulation defect in ljthic. Overexpression of LjTHIC in Lotus japonicus (OE-LjTHIC) decreased shoot growth and increased the activity of the enzymes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. OE-LjTHIC plants exhibited an increase in the number of infection threads and also developed more nodules, which were of smaller size but unchanged nitrogenase activity compared to the wildtype. Taken together, our results suggest that endogenous thiamine produced via LjTHIC acts as an essential nutrient provided by the host plant for rhizobial infection and nodule growth in the Lotus japonicus – rhizobium interaction.

Impaired Pentose Phosphate Pathway in the Spinal Cord of the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Impairments in energy metabolism in amyotrophic lateral sclerosis (ALS) have long been known. However, the changes in the energy-producing pathways in ALS are not comprehensively understood. To investigate specific alterations in glucose metabolism in glycolytic, pentose phosphate, and TCA cycle pathways, we injected uniformly labeled [U-13C]glucose to wild-type and hSOD1G93A mice at symptom onset (80days). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), levels of metabolites were determined in extracts of the cortex and spinal cord. In addition, the activities of several enzymes involved in glucose metabolism were quantified. In the spinal cord, the levels of pentose phosphate pathway (PPP) intermediate ribose 5-phosphate (p = 0.037) were reduced by 37% in hSOD1G93A mice, while the % 13C enrichments in glucose 6-phosphate were increased threefold. The maximal activities of the enzyme glucose 6-phosphate dehydrogenase were decreased by 24% in the spinal cord (p = 0.005), suggesting perturbations in the PPP. The total amount of pyruvate in the cortex (p = 0.039) was reduced by 20% in hSOD1G93A mice. Also, the activities of the glycolytic enzyme pyruvate kinase were reduced in the cortex by 31% (p = 0.002), indicating alterations in glycolysis. No significant differences were seen in the total amounts as well as % 13C enrichments in most TCA cycle intermediates, suggesting largely normal TCA cycle function. On the other hand, oxoglutarate dehydrogenase activity was decreased in the cortex, which may indicate increased oxidative stress. Overall, this study revealed decreased activity of the PPP in the spinal cord and alterations in glycolysis in hSOD1G93A mouse CNS tissues at the early symptomatic stage of disease.

Administration of chitosan-tripolyphosphate-DNA nanoparticles to knockdown glutamate dehydrogenase expression impairs transdeamination and gluconeogenesis in the liver

Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53–55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.

Functional Analysis of Methylomonas sp. DH-1 Genome as a Promising Biocatalyst for Bioconversion of Methane to Valuable Chemicals

Methylomonas sp. DH-1, newly isolated from the activated sludge of a brewery plant, has been used as a promising biocatalytic platform for the conversion of methane to value-added chemicals. Methylomonas sp. DH-1 can efficiently convert methane and propane into methanol and acetone with a specific productivity of 4.31 and 0.14 mmol/g cell/h, the highest values ever reported, respectively. Here, we present the complete genome sequence of Methylomonas sp. DH-1 which consists of a 4.86 Mb chromosome and a 278 kb plasmid. The existence of a set of genes related to one-carbon metabolism and various secondary metabolite biosynthetic pathways including carotenoid pathways were identified. Interestingly, Methylomonas sp. DH-1 possesses not only the genes of the ribulose monophosphate cycle for type I methanotrophs but also the genes of the serine cycle for type II. Methylomonas sp. DH-1 accumulated 80 mM succinate from methane under aerobic conditions, because DH-1 has 2-oxoglutarate dehydrogenase activity and the ability to operate the full TCA cycle. Availability of the complete genome sequence of Methylomonas sp. DH-1 enables further investigations on the metabolic engineering of this strain for the production of value-added chemicals from methane.

Positive correlation between rat brain glutamate concentrations and mitochondrial 2-oxoglutarate dehydrogenase activity

Glutamate is a key metabolite and major excitatory neurotransmitter, degraded through transformation to 2-oxoglutarate which is further catabolized by 2-oxoglutarate dehydrogenase complex (OGDHC). Both the glutamate excitotoxicity and impaired OGDHC activity are hallmarks of neurodegeneration. This work quantifies a relationship between the brain OGDHC activity and glutamate levels, assessing its diagnostic value to characterize (patho)physiology. A moderate to strong positive correlation of the two parameters determined under varied physiological settings (brain regions, seasons, gender, pregnancy, rat line), is revealed. Mitochondrial impairment (OGDHC inhibition or acute hypobaric hypoxia) decreases the interdependence, even when the parameter means do not change significantly. Compared to the cortex, the cerebellum exhibits a lower inter-individual glutamate variation and a weaker glutamate-OGDHC interdependence. Specific metabolism of the brain regions is also characterized by a positive correlation between glutamate and γ-aminobutyric acid (GABA) concentrations in the cortex but not in the cerebellum. In contrast, a strong positive correlation between glutamate and glutamine is present in both the cortex and cerebellum. The differences in metabolic correlations are in line with transcriptomics data which suggest that glutamate distribution between competitive pathways contributes to the brain-region-specific features of the interdependences of glutamate and OGDHC or GABA.

Alterations in Cytosolic and Mitochondrial [U-13C]Glucose Metabolism in a Chronic Epilepsy Mouse Model.

Temporal lobe epilepsy is a common form of adult epilepsy and shows high resistance to treatment. Increasing evidence has suggested that metabolic dysfunction contributes to the development of seizures, with previous studies indicating impairments in brain glucose metabolism. Here we aim to elucidate which pathways involved in glucose metabolism are impaired, by tracing the hippocampal metabolism of injected [U-13C]glucose (i.p.) during the chronic stage of the pilocarpine-status epilepticus mouse model of epilepsy. The enrichment of 13C in the intermediates of glycolysis and the TCA cycle were quantified in hippocampal extracts using liquid chromatography–tandem mass spectroscopy, along with the measurement of the activities of enzymes in each pathway. We show that there is reduced incorporation of 13C in the intermediates of glycolysis, with the percentage enrichment of all downstream intermediates being highly correlated with those of glucose 6-phosphate. Furthermore, the activities of all enzymes in this pathway including hexokinase and phosphofructokinase were unaltered, suggesting that glucose uptake is reduced in this model without further impairments in glycolysis itself. The key findings were 33% and 55% losses in the activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively, along with reduced 13C enrichment in TCA cycle intermediates. This lower 13C enrichment is best explained in part by the reduced enrichment in glycolytic intermediates, whereas the reduction of key TCA cycle enzyme activity indicates that TCA cycling is also impaired in the hippocampal formation. Together, these data suggest that multitarget approaches may be necessary to restore metabolism in the epileptic brain.

Improved fermentative production of gamma-aminobutyric acid via the putrescine route: Systems metabolic engineering for production from glucose, amino sugars, and xylose.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-1 h-1 , the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862-873. © 2016 Wiley Periodicals, Inc.

Ciprofloxacin triggered glutamate production by Corynebacterium glutamicum.

Background Corynebacterium glutamicum is a well-studied bacterium which naturally overproduces glutamate when induced by an elicitor. Glutamate production is accompanied by decreased 2-oxoglutatate dehydrogenase activity. Elicitors of glutamate production by C. glutamicum analyzed to molecular detail target the cell envelope.ResultsCiprofloxacin, an inhibitor of bacterial DNA gyrase and topoisomerase IV, was shown to inhibit growth of C. glutamicum wild type with concomitant excretion of glutamate. Enzyme assays showed that 2-oxoglutarate dehydrogenase activity was decreased due to ciprofloxacin addition. Transcriptome analysis revealed that this inhibitor of DNA gyrase increased RNA levels of genes involved in DNA synthesis, repair and modification. Glutamate production triggered by ciprofloxacin led to glutamate titers of up to 37 ± 1 mM and a substrate specific glutamate yield of 0.13 g/g. Even in the absence of the putative glutamate exporter gene yggB, ciprofloxacin effectively triggered glutamate production. When C. glutamicum wild type was cultivated under nitrogen-limiting conditions, 2-oxoglutarate rather than glutamate was produced as consequence of exposure to ciprofloxacin. Recombinant C. glutamicum strains overproducing lysine, arginine, ornithine, and putrescine, respectively, secreted glutamate instead of the desired amino acid when exposed to ciprofloxacin.ConclusionsCiprofloxacin induced DNA synthesis and repair genes, reduced 2-oxoglutarate dehydrogenase activity and elicited glutamate production by C. glutamicum. Production of 2-oxoglutarate could be triggered by ciprofloxacin under nitrogen-limiting conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0857-6) contains supplementary material, which is available to authorized users.

Специфічність функціонування дегідрогеназ циклу Кребса і антиоксидантних ферментів м’язових тканин гусей в умовах гіпо- та гіпероксії

The processes of energy metabolism are essential for the functioning of all living organisms. However,in redox processes of energy metabolism, in addition to synthesis of the main macroergic compounds produce large quantities of reactive oxygen species, which initiate free-radical processes in cells. Free radical oxidation of lipids is important, as it provide a number of important functions. The series of mutual regulation, in which excessive activation of energy processes leads to intensification of peroxidation and thus inhibits energy processes are included during the physiological functioning of the body.However, the increased production of free radicals that accompanies most ofpathological conditions, leads to a variety of cell damages. Mechanisms of peroxidation and thermal oxidation were investigated enough detail for most organisms, both in physiological conditions and for various pathological conditions. However, the physiological functioning of any organism provides not only a level of redox processes, but also a dynamic balance between their views. Clarification of mechanisms support of this balance will facilitate to right choice of strategies for remedial action. The purpose of the study was to determine changes of activity enzymes of Citric acid cycle and antioxidant protection and the relationships between them in physiologically stressful period of transition from fetal to postnatal existence in muscle tissues of geese. Fabrics of geese are characterized by a high content of substrate of processes oxidation fatty acids and were selected as model species due to the high intensity of their metabolism. Experimentally investigated changes in activity of dehydrogenases Citric acid cycle`s(succinate dehydrogenase, pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, malate dehydrogenase) and antioxidant enzymes (glutathione peroxidase, catalase, superoxide dismutase) in the striated muscles of limb and smooth muscles of the stomach of geese from mid-embryogenesis to 14-day old postnatal period.Defining the nature of the relationship of the Krebs cycle dehydrogenases and antioxidant enzymes as within each of the systems, and as between them was conducted using correlation analysis. It is established that rapid activation during embryogenesis and then braking during the transition to postnatal life is specific for Krebs cycle dehydrogenases of striated muscles.Formation of adaptive responses to new living conditions from the 7th to the 14th day of postnatal period is accompanied by activation of Krebs cycle dehydrogenases to the maximum level.Activation of dehydrogenases in the smooth muscles of the stomach of geese occurs more slowly, but the transition to postnatal existence does not causereduction of their activity.However, the maximum level of activity of Krebs cycle dehydrogenases in the stomach of geese are also installed during postnatal development.In striated muscles the highest level of activity of all studied antioxidant enzymes was observed in the embryonic period. In the stomach activity of superoxide dismutase and glutathione peroxidase reach a maximum after hatching goslings in the postnatal period.The results of correlative analysis of activity of succinate dehydrogenase, pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, malate dehydrogenase in striated muscles show enough high level of consistency within this system.However, the same level of relations established between the activity of Krebs cycle dehydrogenases and antioxidant enzymes: pyruvate dehydrogenase and glutathione peroxidase, succinate dehydrogenase and catalase and somewhat weaker between 2-oxoglutarate dehydrogenase and catalase. In the stomach from all possible significant correlations is only one - between of activity of 2-oxoglutarate dehydrogenase and catalase. Thus, tissue specificity of functioning of dehydrogenases cycle Citric acid in striated muscles is their more rapid activation during embryogenesis, braking activity during the transition to postnatal existence and further intensification from 7 days against the background of the formation of an adaptive response to new conditions. Activation of all dehydrogenases in the stomach occurs more slowly and reaches its maximum level already in the postnatal period. Maintaining the balance of biological oxidation and peroxidation in examined tissues is realized through mutual regulation of enzyme activities antioxidant system and energy metabolism, about that evidenced a close correlation between these indicators.