Α-ketoglutarate Dehydrogenase Complex Research Articles

The Impact of Thiamine Treatment in the Diabetes Mellitus

Thiamine acts as a coenzyme for transketolase (Tk) and for the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes, enzymes which play a fundamental role for intracellular glucose metabolism. The relationship between thiamine and diabetes mellitus (DM) has been reported in the literature. Thiamine levels and thiamine-dependent enzyme activities have been reduced in DM. Genetic studies provide opportunity to link the relationship between thiamine and DM (such as Tk, SLC19A2 gene, transcription factor Sp1, α-1-antitrypsin, and p53). Thiamine and its derivatives have been demonstrated to prevent the activation of the biochemical pathways (increased flux through the polyol pathway, formation of advanced glycation end-products, activation of protein kinase C, and increased flux through the hexosamine biosynthesis pathway) induced by hyperglycemia in DM.Thiamine definitively has a role in the diabetic endothelial vascular diseases (micro and macroangiopathy), lipid profile, retinopathy, nephropathy, cardiopathy, and neuropathy.

Kinetic Modeling of the Mitochondrial Energy Metabolism of Neuronal Cells: The Impact of Reducedα-Ketoglutarate Dehydrogenase Activities on ATP Production and Generation of Reactive Oxygen Species

Reduced activity of brain α-ketoglutarate dehydrogenase complex (KGDHC) occurs in a number of neurodegenerative diseases like Parkinson's disease and Alzheimer's disease. In order to quantify the relation between diminished KGDHC activity and the mitochondrial ATP generation, redox state, transmembrane potential, and generation of reactive oxygen species (ROS) by the respiratory chain (RC), we developed a detailed kinetic model. Model simulations revealed a threshold-like decline of the ATP production rate at about 60% inhibition of KGDHC accompanied by a significant increase of the mitochondrial membrane potential. By contrast, progressive inhibition of the enzyme aconitase had only little impact on these mitochondrial parameters. As KGDHC is susceptible to ROS-dependent inactivation, we also investigated the reduction state of those sites of the RC proposed to be involved in ROS production. The reduction state of all sites except one decreased with increasing degree of KGDHC inhibition suggesting an ROS-reducing effect of KGDHC inhibition. Our model underpins the important role of reduced KGDHC activity in the energetic breakdown of neuronal cells during development of neurodegenerative diseases.

Deficits in the mitochondrial enzyme α-ketoglutarate dehydrogenase lead to Alzheimer's disease-like calcium dysregulation

Understanding the molecular sequence of events that culminate in multiple abnormalities in brains from patients that died with Alzheimer's disease (AD) will help to reveal the mechanisms of the disease and identify upstream events as therapeutic targets. The activity of the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) in homogenates from autopsy brain declines with AD. Experimental reductions in KGDHC in mouse models of AD promote plaque and tangle formation, the hallmark pathologies of AD. We hypothesize that deficits in KGDHC also lead to the abnormalities in endoplasmic reticulum (ER) calcium stores and cytosolic calcium following K+ depolarization that occurs in cells from AD patients and transgenic models of AD. The activity of the mitochondrial enzyme KGDHC was diminished acutely (minutes), long-term (days), or chronically (weeks). Acute inhibition of KGDHC produced effects on calcium opposite to those in AD, while the chronic or long-term inhibition of KGDHC mimicked the AD-related changes in calcium. Divergent changes in proteins released from the mitochondria that affect endoplasmic reticulum calcium channels may underlie the selective cellular consequences of acute versus longer term inhibition of KGDHC. The results suggest that the mitochondrial abnormalities in AD can be upstream of those in calcium.

Mutations in the Dimer Interface of Dihydrolipoamide Dehydrogenase Promote Site-specific Oxidative Damages in Yeast and Human Cells

Dihydrolipoamide dehydrogenase (DLD) is a multifunctional protein well characterized as the E3 component of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes. Previously, conditions predicted to destabilize the DLD dimer revealed that DLD could also function as a diaphorase and serine protease. However, the relevance of these cryptic activities remained undefined. We analyzed human DLD mutations linked to strikingly different clinical phenotypes, including E340K, D444V, R447G, and R460G in the dimer interface domain that are responsible for severe multisystem disorders of infancy and G194C in the NAD(+)-binding domain that is typically associated with milder presentations. In vitro, all of these mutations decreased to various degrees dihydrolipoamide dehydrogenase activity, whereas dimer interface mutations also enhanced proteolytic and/or diaphorase activity. Human DLD proteins carrying each individual mutation complemented fully the respiratory-deficient phenotype of yeast cells lacking endogenous DLD even when residual dihydrolipoamide dehydrogenase activity was as low as 21% of controls. However, under elevated oxidative stress, expression of DLD proteins with dimer interface mutations greatly accelerated the loss of respiratory function, resulting from enhanced oxidative damage to the lipoic acid cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase and other mitochondrial targets. This effect was not observed with the G194C mutation or a mutation that disrupts the proteolytic active site of DLD. As in yeast, lipoic acid cofactor was damaged in human D444V-homozygous fibroblasts after exposure to oxidative stress. We conclude that the cryptic activities of DLD promote oxidative damage to neighboring molecules and thus contribute to the clinical severity of DLD mutations.

Detailed kinetics and regulation of mammalian 2-oxoglutarate dehydrogenase

BackgroundMitochondrial 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex (OGDHC), a key regulatory point of tricarboxylic acid (TCA) cycle, plays vital roles in multiple pathways of energy metabolism and biosynthesis. The catalytic mechanism and allosteric regulation of this large enzyme complex are not fully understood. Here computer simulation is used to test possible catalytic mechanisms and mechanisms of allosteric regulation of the enzyme by nucleotides (ATP, ADP), pH, and metal ion cofactors (Ca2+ and Mg2+).ResultsA model was developed based on an ordered ter-ter enzyme kinetic mechanism combined with con-formational changes that involve rotation of one lipoic acid between three catalytic sites inside the enzyme complex. The model was parameterized using a large number of kinetic data sets on the activity of OGDHC, and validated by comparison of model predictions to independent data.ConclusionsThe developed model suggests a hybrid rapid-equilibrium ping-pong random mechanism for the kinetics of OGDHC, consistent with previously reported mechanisms, and accurately describes the experimentally observed regulatory effects of cofactors on the OGDHC activity. This analysis provides a single consistent theoretical explanation for a number of apparently contradictory results on the roles of phosphorylation potential, NAD (H) oxidation-reduction state ratio, as well as the regulatory effects of metal ions on ODGHC function.

Functional Plasticity and Allosteric Regulation of α-Ketoglutarate Decarboxylase in Central Mycobacterial Metabolism

The α-ketoglutarate dehydrogenase (KDH) complex is a major regulatory point of aerobic energy metabolism. Mycobacterium tuberculosis was reported to lack KDH activity, and the putative KDH E1o component, α-ketoglutarate decarboxylase (KGD), was instead assigned as a decarboxylase or carboligase. Here, we show that this protein does in fact sustain KDH activity, as well as the additional two reactions, and these multifunctional properties are shared by the Escherichia coli homolog, SucA. We also show that the mycobacterial enzyme is finely regulated by an additional acyltransferase-like domain and bythe action of acetyl-CoA, a powerful allosteric activator able to enhance the concerted protein motions observed during catalysis. Our results uncover the functional plasticity of a crucial node in bacterial metabolism, which may be important for M. tuberculosis during host infection.

Multiple Targets of Nitric Oxide in the Tricarboxylic Acid Cycle of Salmonella enterica Serovar Typhimurium

Host nitric oxide (NO⋅) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NO⋅-induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NO⋅ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO⋅ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NO⋅-producing mice, demonstrating that NO⋅-induced MK auxotrophy occurs invivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NO⋅.

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg⁻¹ protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding α-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway.

Inactivation and Reactivation of the Mitochondrial α-Ketoglutarate Dehydrogenase Complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.

Brain [U‐13C]glucose metabolism in mice with decreased α‐ketoglutarate dehydrogenase complex activity

The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), a mitochondrial enzyme complex that mediates the oxidative decarboxylation of α-ketoglutarate in the TCA cycle, is reduced in Alzheimer's disease. We investigated the metabolic effects of a partial KGDHC activity reduction on brain glucose metabolism using mice with disrupted expression of dihydrolipoyl succinyltransferase (DLST; gene encoding the E2k subunit of KGDHC). Brain tissue extracts from cortex and cerebellum of 6-week-old heterozygote DLST knockout mice (DLST+/-) and corresponding wild-type mice injected with [U-(13) C]glucose and decapitated 15 min later were analyzed. An increase in the concentration of glucose in cortex suggested a decrease in the cortical utilization of glucose in DLST+/- mice. Furthermore, the concentration and (13) C labelling of aspartate in cortex were reduced in DLST+/- mice. This decline was likely caused by a decrease in the pool of oxaloacetate. In contrast to results from cell culture studies, no indications of altered glycolysis or GABA shunt activity were found. Glucose metabolism in the cerebellum was unaffected by the decrease in KGDHC activity. Among metabolites not related to glucose metabolism, the concentration of taurine was decreased in the cortex, and that of tyrosine was increased in the cerebellum. These results imply that diminished KGDHC activity has the potential to induce the reduction in glucose utilization that is seen in several neurodegenerative diseases.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Succinylphosphonate Esters Are Competitive Inhibitors of MenD That Show Active-Site Discrimination between Homologous α-Ketoglutarate-Decarboxylating Enzymes

MenD is a thiamin diphosphate-dependent enzyme catalyzing the first unique step in menaquinone biosynthesis in bacteria. We have synthesized acylphosphonate ester analogues of alpha-ketoglutarate, a substrate of MenD. These compounds are competitive inhibitors of MenD, with K(i) values as low as 700 nM. Observed structure-activity relationships are in notable contrast to those reported previously for succinylphosphonate inhibition of the alpha-ketoglutarate dehydrogenase complex, despite the apparent homology of these enzymes, and the identical decarboxylation reactions catalyzed. Inhibiting menaquinone biosynthesis is a proposed approach to inhibiting Mycobacterium tuberculosis growth. These inhibitors showed no significant inhibition of M. tuberculosis growth in vitro under aerobic and hypoxic conditions but give new information about the binding characteristics of the MenD active site. Site-directed mutation of Ser391 to alanine had only a minor effect on catalysis, but even the conservative mutation of Arg395 to lysine had a significant effect on the catalytic processing of isochorismate.

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; An unexpected effect of cardiolipin in α-ketoglutarate dehydrogenase and prohibitin complexes

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Delta strain; taz1Delta strain; and acb1Deltataz1Delta strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains. Furthermore, a TAZ1ACB1 double deletion mutant strain was used in this study which has both a decrease in the length of the CL acyl chains and an increase in MLCL. BN/SDS PAGE analysis revealed that cardiolipin is important for the prohibitin-m-AAA protease complex, the alpha-ketoglutarate dehydrogenase complex and respiratory chain supercomplexes. The results indicate that the decreased level of complexes in taz1Delta and acb1Deltataz1Delta mitochondria is due to a decreased content of CL or the presence of MLCL.

Mice deficient in dihydrolipoyl succinyl transferase show increased vulnerability to mitochondrial toxins

The activity of a key mitochondrial tricarboxylic acid cycle enzyme, α-ketoglutarate dehydrogenase complex (KGDHC), declines in many neurodegenerative diseases. KGDHC consists of three subunits. The dihydrolipoyl succinyl transferase (DLST) component is unique to KGDHC. DLST +/− mice showed reduced mRNA and protein levels and decreased brain mitochondrial KGDHC activity. Neurotoxic effects of mitochondrial toxins were exacerbated in DLST +/− mice. MPTP produced a significantly greater reduction of striatal dopamine and tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta of DLST +/− mice. DLST deficiency enhanced the severity of lipid peroxidation in the substantia nigra after MPTP treatment. Striatal lesions induced by either malonate or 3-nitropropionic acid (3-NP) were significantly larger in DLST +/− mice than in wildtype controls. DLST deficiency enhanced the 3-NP inhibition of mitochondria enzymes, and 3-NP induced protein and DNA oxidations. These observations support the hypothesis that reductions in KGDHC may impair the adaptability of the brain and contribute to the pathogenesis of neurodegenerative diseases.

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.

Enhancement of α-ketoglutarate production in Torulopsis glabrata: Redistribution of carbon flux from pyruvate to α-ketoglutarate

This manuscript aimed at increasing the production of α-ketoglutarate by the multi-vitamin auxotrophic yeast Torulopsis glabrata CCTCC M202019. The carbon flux was redistributed from pyruvate to α-ketoglutarate node by manipulating the specific activity of pyruvate dehydrogenase complex (PDH), pyruvate carboxylase (PC), pyruvate decarboxylase (PDC), and α-ketoglutarate dehydrogenase complex (KGDH). By proper increase of PDH, PC, and PDC activities, α-ketoglutarate in fermentation broth could be accumulated to the levels of 17.1 g/L, 21.6 g/L, and 31.2 g/L, respectively. In addition, decrease in the specific activity of KGDH also resulted in an enhanced α-ketoglutarate synthesis. With a proposed combinational enzymes regulation strategy, the highest α-ketoglutarate concentration of 37.7 g/L was achieved.

P.2.06 Enhancement of classical conditioning and increase in neurogenesis by neuronal disruption of glyT1 in the forebrain

Mitochondrial defects including reduction of a key mitochondrial tricarboxylic acid cycle enzyme α-ketoglutarate–dehydrogenase complex (KGDHC) are characteristic of many neurodegenerative diseases. KGDHC consists of α-ketoglutarate dehydrogenase, dihydrolipoyl succinyltransferase (E2k), and dihydrolipoamide dehydrogenase (Dld) subunits. We investigated whether Dld or E2k deficiency influences adult brain neurogenesis using immunohistochemistry for the immature neuron markers, doublecortin (Dcx) and polysialic acid–neural cell adhesion molecule, as well as a marker for proliferation, proliferating cell nuclear antigen (PCNA). Both Dld- and E2k-deficient mice showed reduced Dcx-positive neuroblasts in the subgranular zone (SGZ) of the hippocampal dentate gyrus compared with wild-type mice. In the E2k knockout mice, increased immunoreactivity for the lipid peroxidation marker, malondialdehyde occurred in the SGZ. These alterations did not occur in the subventricular zone (SVZ). PCNA staining revealed decreased proliferation in the SGZ of E2k-deficient mice. In a transgenic mouse model of Alzheimer's disease, Dcx-positive cells in the SGZ were also reduced compared with wild type, but Dld deficiency did not exacerbate the reduction. In the malonate lesion model of Huntington's disease, Dld deficiency did not alter the lesion-induced increase and migration of Dcx-positive cells from the SVZ into the ipsilateral striatum. Thus, the KGDHC subunit deficiencies associated with elevated lipid peroxidation selectively reduced the number of neuroblasts and proliferating cells in the hippocampal neurogenic zone. However, these mitochondrial defects neither exacerbated certain pathological conditions, such as amyloid precursor protein (APP) mutation-induced reduction of SGZ neuroblasts, nor inhibited malonate-induced migration of SVZ neuroblasts. Our findings support the view that mitochondrial dysfunction can influence the number of neural progenitor cells in the hippocampus of adult mice.

Inhibition by methylated organoarsenicals of the respiratory 2-oxo-acid dehydrogenases

Inorganic arsenic that is ingested through drinking water or inhalation is metabolized by biological methylation pathways into organoarsenical metabolites. It is now becoming understood that this metabolism that was formerly considered to be detoxification may contribute as much or more to increasing the toxicity of arsenic. One proposed mode of the toxic action of arsenic and its organoarsenic metabolites is through its binding to proteins and inactivating their enzymatic activity. The classic case has been considered the affinity of the proximal 1,3 sulfhydryl groups of the lipoic acid cofactor of the pyruvate dehydrogenase complex for arsenic. A 2:1 stoichiometry of sulfhydryl to arsenic groups has been measured in proteins and arsenical complexes can be synthesized using free d, l-lipoic acid. The relative importance of this site for arsenic binding has come in to question through the use of methylating bifunctional arsenic complexes, and the suggestion that arsenic inhibits the pyruvate dehydrogenase complex indirectly by elevating mitochondrial hydrogen peroxide generation. In order to separate the effects of direct trivalent arsenite toxicity from that of hydrogen peroxide and activated oxygen, we studied the inhibition of the PDH complex under conditions that did not generate hydrogen peroxide but did expose the lipoic acid group in its reduced state to arsenicals. We also studied the effects of arsenicals in the inhibition of the α-ketoglutarate dehydrogenase complex. We found that only trivalent arsenical compounds inhibited the activity of both dehydrogenase complexes and only when the lipoic acid was in its reduced form. Arsenite inhibited both enzyme complexes approximately equivalently while monomethylarsenite inhibited the PDH complex to a greater extent than the KGDH complex – although both complexes were very sensitive to inhibition by this complex. Dimethylarsenite inhibition of both complexes was only observed with longer pre-incubation periods. Cumulative inhibition by the reduced arsenical was observed for all complexes indicating a binding mode of inhibition that is dependent upon lipoic acid being in its reduced state.

Characterization of Two Naturally Occurring Mutations Close to Cofactors in Human Dihydrolipoamide Dehydrogenase

Dihydrolipoamide dehydrogenase (E3) (dihydrolipoamide: NAD oxidoreductase; EC 1.8.1.4) exists as a common component in three α-keto acid dehydrogenase complexes (pyruvate, α-ketoglutarate and branched-chain α-keto acid dehydrogenase complexes) and the glycine cleavage system. It catalyzes the reoxidation of the dihydrolipoyl group of the acyltransferase components of the α-keto acid dehydrogenase complexes and the reoxidation of that of the hydrogen-carrier protein of the glycine cleavage system. It is a homodimeric enzyme containing one FAD as a prosthetic group at each subunit. Two naturally occurring mutations near cofactors in human E3 were reported. One mutation was from E3 deficient patients of Ashkenazi Jewish. A common mutation of Gly-194 to Cys was found in their E3 sequences. Their clinical symptoms were the recurrent attacks of vomiting, abdominal pain, neonatal suffering, muscle weakness and exertional fatigue. Another mutation in a boy having reduced E3 activity was also reported. At the age of ten weeks, his clinical symptoms were vomiting, progressive hypotonia and a severe lactic acidosis. He possessed a mutation of Met-326 to Val in his E3 sequence. Site-directed mutagenesis method has been a useful tool for the structure-function study of human E3 and other proteins. By the site-specific mutations of Gly-194 to Cys and Met-326 to Val, the effects of these naturally occurring mutations close to cofactors in human E3 structure and function were examined. Site-directed mutagenesis was performed using a mutagenesis kit according to the provided protocol. PCRs were carried out using the human E3 expression vector pPROEX-1:E3 as a template in a programmable PCR machine with the appropriate primers. Whole DNA sequence of human E3 coding region was sequenced to verify the integrity of DNA sequences and the mutations. Purification of the Val-326 mutant was unsuccessful. There was not enough of the Val-326 mutant present to be purified when its expression was induced in E. coli. The SDS-PAGE gel showed that very small amounts of the mutant were present in the soluble fraction. This indicated that the mutant could have problems in its structural stability and/or proper folding processes while it was expressed in E. coli. The Met-326 is located very close to both FAD and NAD in the E3 structure as shown in Figure 1A and involved in several direct interactions with them. The substitution of the Met-326 with smaller Val probably causes structural changes that affect more directly the bindings of near FAD and NAD to the enzyme (Figure 1B). These structural changes could be detrimental enough to destroy the stable expression of the enzyme in E. coli. The Cys-194 mutant was expressed in E. coli enough to be purified and characterized even though the amounts of purified enzyme were about 12-fold lesser than those of normal enzyme. The purification of the Cys-194 mutant was performed using nickel affinity column according to the provided protocol. SDS-PAGE gel showed that the mutant was highly purified. E3 assay was performed at 37 C in a 50 mM potassium phosphate buffer (pH 8.0) containing 1.5

α-Ketoglutarate dehydrogenase contributes to production of reactive oxygen species in glutamate-stimulated hippocampal neurons in situ

The α-ketoglutarate dehydrogenase complex (KGDHC) which catalyzes the conversion of α-ketoglutarate to succinyl-CoA and NADH in mitochondria, is known to generate O2·− in vitro. To find out if KGDHC contributes to neuronal reactive oxygen species (ROS) increase in situ, we investigated whether the specific inhibitors of cellular KGDHC, succinyl phosphonate (SP) and the SP triethyl ester (TESP), might affect the glutamate-induced ROS production in cultured hippocampal neurons from rats. The concentration-dependent decrease in the mitochondrial potential of the glutamate-overstimulated neurons in the presence of SP or TESP indicated that under the conditions inducing neuronal ROS generation, the inhibitors are delivered to mitochondria, and their subsequent inhibition of KGDHC decreases the mitochondrial potential. The production of O2·− was detected by reaction with hydroethidine. The distribution of the resulting fluorescence of DNA-ethidium coincided with that of the mitochondrial marker Mitotracker, pointing to the mitochondrial origin of the hydroethidine-detected ROS in response to glutamate (100 μM). At 200 μM, both TESP and SP administered together with glutamate, inhibited the glutamate-induced ROS production by about 20%, with the inhibition increasing to 44% at 500 μM TESP. The decrease in neuronal ROS by specific inhibitors of KGDHC demonstrates that KGDHC is a source of ROS in cultured neurons responding to glutamate. However, increasing the concentration of the strongest KGDHC inhibitor SP to 500 μM even increased the ROS production compared with glutamate alone, presumably due to secondary effects arising upon the strong KGDHC inhibition. Our work extends the current understanding of the glutamate-induced ROS generation in neurons, shedding light on the pathological mechanisms of the KGDHC involvement in glutamate neurotoxicity. In conclusion, potent KGDHC inhibitors are promising diagnostic tools for in situ study of neurodegenerative mechanisms.