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Frank Jordan - One of the best experts on this subject based on the ideXlab platform.
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human 2 Oxoglutarate Dehydrogenase and 2 oxoadipate Dehydrogenase both generate superoxide h2o2 in a side reaction and each could contribute to oxidative stress in mitochondria
Neurochemical Research, 2019Co-Authors: Frank Jordan, Natalia Nemeria, Gary GerfenAbstract:According to recent findings, the human 2-Oxoglutarate Dehydrogenase Complex (hOGDHc) could be an important source of the reactive oxygen species in the mitochondria and could contribute to mitochondrial abnormalities associated with multiple neurodegenerative diseases, including Alzheimer’s disease, Huntington disease, and Parkinson’s disease. The human 2-oxoadipate Dehydrogenase (hE1a) is a novel protein, which is encoded by the DHTKD1 gene. Both missence and nonsense mutations were identified in the DHTKD1 that lead to alpha-aminoadipic and alpha-oxoadipic aciduria, a metabolic disorder with a wide variety of the neurological abnormalities, and Charcot-Marie-Tooth disease type 2Q, an inherited neurological disorder affecting the peripheral nervous system. Recently, the rare pathogenic mutations in DHTKD1 and an increased H2O2 production were linked to the genetic ethiology of Eosinophilic Esophagitis (EoE), a chronic allergic inflammatory esophageal disorder. In view of the importance of hOGDHc in the tricarboxylic acid cycle (TCA cycle) and hE1a on the l-lysine, l-hydroxylysine and l-tryptophan degradation pathway in mitochondria, and to enhance our current understanding of the mechanism of superoxide/H2O2 generation by hOGDHc, and by human 2-oxoadipate Dehydrogenase Complex (hOADHc), this review focuses on several novel and unanticipated recent findings in vitro that emerged from the Jordan group’s research. Most significantly, the hE1o and hE1a now join the hE3 as being able to generate the superoxide/H2O2 in mitochondria.
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evidence for functional and regulatory cross talk between the tricarboxylic acid cycle 2 Oxoglutarate Dehydrogenase Complex and 2 oxoadipate Dehydrogenase on the l lysine l hydroxylysine and l tryptophan degradation pathways from studies in vitro
Biochimica et Biophysica Acta, 2018Co-Authors: Natalia S Nemeria, Luying Yang, Gary J Gerfen, Xu Zhang, Frank JordanAbstract:Abstract Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-Oxoglutarate Dehydrogenase Complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate Dehydrogenase Complex (hOADHc) from the final degradation pathway of l -lysine, l -hydroxylysine and l -tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (kcat/Km) were displayed by the two Complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl Dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subComplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both Complexes produced superoxide/H2O2 from O2 in the reductive half reaction suggesting that hE1o, and hE1a (within their Complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-Oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two Complexes on the different metabolic pathways.
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the mitochondrial 2 oxoadipate and 2 Oxoglutarate Dehydrogenase Complexes share their e2 and e3 components for their function and both generate reactive oxygen species
Free Radical Biology and Medicine, 2018Co-Authors: Natalia S Nemeria, Pradeep Nareddy, Luying Yang, Gary J Gerfen, Michal Szostak, Xu Zhang, Frank JordanAbstract:Abstract Herein are reported unique properties of the novel human thiamin diphosphate (ThDP)-dependent enzyme 2-oxoadipate Dehydrogenase (hE1a), known as Dehydrogenase E1 and transketolase domain-containing protein 1 that is encoded by the DHTKD1 gene. It is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA on the final degradative pathway of L -lysine and is critical for mitochondrial metabolism. Functionally active recombinant hE1a has been produced according to both kinetic and spectroscopic criteria in our toolbox leading to the following conclusions: (i) The hE1a has recruited the dihydrolipoyl succinyltransferase (hE2o) and the dihydrolipoyl Dehydrogenase (hE3) components of the tricarboxylic acid cycle 2-Oxoglutarate Dehydrogenase Complex (OGDHc) for its activity. (ii) 2-Oxoglutarate (OG) and 2-oxoadipate (OA) could be oxidized by hE1a, however, hE1a displays an approximately 49-fold preference in catalytic efficiency for OA over OG, indicating that hE1a is specific to the 2-oxoadipate Dehydrogenase Complex. (iii) The hE1a forms the ThDP-enamine radical from OA according to electron paramagnetic resonance detection in the oxidative half reaction, and could produce superoxide and H2O2 from decarboxylation of OA in the forward physiological direction, as also seen with the 2-Oxoglutarate Dehydrogenase hE1o component. (iv) Once assembled to Complex with the same hE2o and hE3 components, the hE1o and hE1a display strikingly different regulation: both succinyl-CoA and glutaryl-CoA significantly reduced the hE1o activity, but not the activity of hE1a.
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the human krebs cycle 2 Oxoglutarate Dehydrogenase Complex creates an additional source of superoxide hydrogen peroxide from 2 oxoadipate as alternative substrate
Free Radical Biology and Medicine, 2017Co-Authors: Natalia S Nemeria, Pradeep Nareddy, Elena Guevara, Gary J Gerfen, Michal Szostak, Frank JordanAbstract:Recently, we reported that the human 2-Oxoglutarate Dehydrogenase (hE1o) component of the 2-Oxoglutarate Dehydrogenase Complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-Oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18]]. We here report that hE1o can also utilize the next higher homologue of OG, 2-oxoadipate (OA) as a substrate according to multiple criteria in our toolbox: (i) Both E1o-specific and overall Complex activities (NADH production) were detected using OA as a substrate; (ii) Two post-decarboxylation intermediates were formed by hE1o from OA, the ThDP-enamine and the C2α-hydroxyalkyl-ThDP, with nearly identical rates for OG and OA; (iii) Both OG and OA could reductively acylate lipoyl domain created from dihydrolipoyl succinyltransferase (E2o); (iv) Both OG and OA gave α-ketol carboligaton products with glyoxylate, but with opposite chirality; a finding that could be of utility in chiral synthesis; (v) Dioxygen could oxidize the ThDP-derived enamine from both OG and OA, leading to ThDP-enamine radical and generation of superoxide and H2O2. While the observed oxidation-reduction with dioxygen is only a side reaction of the predominant physiological product glutaryl-CoA, the efficiency of superoxide/ H2O2 production was 7-times larger from OA than from OG, making the reaction of OGDHc with OA one of the important superoxide/ H2O2 producers among 2-oxo acid Dehydrogenase Complexes in mitochondria.
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human 2 Oxoglutarate Dehydrogenase Complex e1 component forms a thiamin derived radical by aerobic oxidation of the enamine intermediate
Journal of Biological Chemistry, 2014Co-Authors: Natalia S Nemeria, Hetalben Patel, Laszlo Tretter, Attila Ambrus, Jieyu Zhou, Gary J Gerfen, Vera Adamvizi, Junjie Wang, Frank JordanAbstract:Abstract Herein are reported unique properties of the human 2-Oxoglutarate Dehydrogenase multienzyme Complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-Oxoglutarate Dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-Oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall Complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.
Victoria I. Bunik - One of the best experts on this subject based on the ideXlab platform.
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conformational transitions in the active site of mycobacterial 2 Oxoglutarate Dehydrogenase upon binding phosphonate analogues of 2 Oxoglutarate from a michaelis like Complex to thdp adducts
Journal of Structural Biology, 2019Co-Authors: Tristan Wagner, Victoria I. Bunik, Alexandra Boyko, Pedro M Alzari, Marco BellinzoniAbstract:Abstract Mycobacterial KGD, the thiamine diphosphate (ThDP)-dependent E1o component of the 2-Oxoglutarate Dehydrogenase Complex (OGDHC), is known to undergo significant conformational changes during catalysis with two distinct conformational states, previously named as the early and late state. In this work, we employ two phosphonate analogues of 2-Oxoglutarate (OG), i.e. succinyl phosphonate (SP) and phosphono ethyl succinyl phosphonate (PESP), as tools to isolate the first catalytic steps and understand the significance of conformational transitions for the enzyme regulation. The kinetics showed a more efficient inhibition of mycobacterial E1o by SP (Ki 0.043 ± 0.013 mM) than PESP (Ki 0.88 ± 0.28 mM), consistent with the different circular dichroism spectra of the corresponding Complexes. PESP allowed us to get crystallographic snapshots of the Michaelis-like Complex, the first one for 2-oxo acid Dehydrogenases, followed by the covalent adduction of the inhibitor to ThDP, mimicking the pre-decarboxylation Complex. In addition, covalent ThDP-phosphonate Complexes obtained with both compounds by co-crystallization were in the late conformational state, probably corresponding to slowly dissociating enzyme-inhibitor Complexes. We discuss the relevance of these findings in terms of regulatory features of the mycobacterial E1o enzymes, and in the perspective of developing tools for species-specific metabolic regulation.
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positive correlation between rat brain glutamate concentrations and mitochondrial 2 Oxoglutarate Dehydrogenase activity
Analytical Biochemistry, 2018Co-Authors: Garik Mkrtchyan, A V Graf, L. K. Trofimova, Alexander L. Ksenofontov, Liudmila A Baratova, Victoria I. BunikAbstract:Abstract 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.
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on the role of the mitochondrial 2 Oxoglutarate Dehydrogenase Complex in amino acid metabolism
Amino Acids, 2013Co-Authors: Wagner L Araujo, Garik Mkrtchyan, A V Graf, L. K. Trofimova, Alisdair R Fernie, Dirk Steinhauser, Leonard Krall, Victoria I. BunikAbstract:Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-Oxoglutarate Dehydrogenase Complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-Oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-Oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-Oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-Oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.
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Phosphonate analogs of 2-Oxoglutarate perturb metabolism and gene expression in illuminated Arabidopsis leaves
Frontiers Media S.A., 2012Co-Authors: Wagner L Araujo, Victoria I. Bunik, Takayuki L. Tohge, Adriano Enunes-nesi, Danilo Menezes Daloso, Mhairi Enimick, Ina Ekrahnert, Greg Emoorhead, Alisdair EfernieAbstract:Although the role of the 2-Oxoglutarate Dehydrogenase Complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf discs with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-OGDHC. Our results indicate that the reduced respiration rates are associated with changes in the regulation of metabolic and signalling pathways leading to an imbalance in carbon-nitrogen metabolism and cell homeostasis. The inducible alteration of primary metabolism was associated with altered expression of genes belonging to networks of amino acids, plant respiration and sugar metabolism. In addition, by using isothermal titration calorimetry we excluded the possibility that the changes in gene expression resulted from an effect on 2OG binding to the carbon/ATP sensing protein PII. We also demonstrated that the 2-Oxoglutarate (2OG) degradation by the 2-Oxoglutarate Dehydrogenase strongly influences the distribution of intermediates of the tricarboxylic acid (TCA) cycle and the GABA shunt. Our results indicate that the TCA cycle activity is clearly working in a non-cyclic manner upon 2-OGDHC inhibition during the light period
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chronic alcoholism in rats induces a compensatory response preserving brain thiamine diphosphate but the brain 2 oxo acid Dehydrogenases are inactivated despite unchanged coenzyme levels
Journal of Neurochemistry, 2011Co-Authors: Yulia Parkhomenko, Pavel Kudryavtsev, Svetlana Yu Pylypchuk, Lilia I Chekhivska, Svetlana P Stepanenko, Andrej A Sergiichuk, Victoria I. BunikAbstract:Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-Oxoglutarate and pyruvate Dehydrogenase Complexes decreased in alcoholic brain. The inactivation of pyruvate Dehydrogenase Complex was caused by its increased phosphorylation. The inactivation of 2-Oxoglutarate Dehydrogenase Complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate Dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.
Natalia S Nemeria - One of the best experts on this subject based on the ideXlab platform.
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evidence for functional and regulatory cross talk between the tricarboxylic acid cycle 2 Oxoglutarate Dehydrogenase Complex and 2 oxoadipate Dehydrogenase on the l lysine l hydroxylysine and l tryptophan degradation pathways from studies in vitro
Biochimica et Biophysica Acta, 2018Co-Authors: Natalia S Nemeria, Luying Yang, Gary J Gerfen, Xu Zhang, Frank JordanAbstract:Abstract Herein are reported findings in vitro suggesting both functional and regulatory cross-talk between the human 2-Oxoglutarate Dehydrogenase Complex (hOGDHc), a key regulatory enzyme within the tricarboxylic acid cycle (TCA cycle), and a novel 2-oxoadipate Dehydrogenase Complex (hOADHc) from the final degradation pathway of l -lysine, l -hydroxylysine and l -tryptophan. The following could be concluded from our studies by using hOGDHc and hOADHc assembled from their individually expressed components in vitro: (i) Different substrate preferences (kcat/Km) were displayed by the two Complexes even though they share the same dihydrolipoyl succinyltransferase (hE2o) and dihydrolipoyl Dehydrogenase (hE3) components; (ii) Different binding modes were in evidence for the binary hE1o-hE2o and hE1a-hE2o subComplexes according to fluorescence titrations using site-specifically labeled hE2o-derived proteins; (iii) Similarly to hE1o, the hE1a also forms the ThDP-enamine radical from 2-oxoadipate (electron paramagnetic resonance detection) in the oxidative half reaction; (iv) Both Complexes produced superoxide/H2O2 from O2 in the reductive half reaction suggesting that hE1o, and hE1a (within their Complexes) could both be sources of reactive oxygen species generation in mitochondria from 2-Oxoglutarate and 2-oxoadipate, respectively; (v) Based on our findings, we speculate that hE2o can serve as a trans-glutarylase, in addition to being a trans-succinylase, a role suggested by others; (vi) The glutaryl-CoA produced by hOADHc inhibits hE1o, as does succinyl-CoA, suggesting a regulatory cross-talk between the two Complexes on the different metabolic pathways.
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the mitochondrial 2 oxoadipate and 2 Oxoglutarate Dehydrogenase Complexes share their e2 and e3 components for their function and both generate reactive oxygen species
Free Radical Biology and Medicine, 2018Co-Authors: Natalia S Nemeria, Pradeep Nareddy, Luying Yang, Gary J Gerfen, Michal Szostak, Xu Zhang, Frank JordanAbstract:Abstract Herein are reported unique properties of the novel human thiamin diphosphate (ThDP)-dependent enzyme 2-oxoadipate Dehydrogenase (hE1a), known as Dehydrogenase E1 and transketolase domain-containing protein 1 that is encoded by the DHTKD1 gene. It is involved in the oxidative decarboxylation of 2-oxoadipate (OA) to glutaryl-CoA on the final degradative pathway of L -lysine and is critical for mitochondrial metabolism. Functionally active recombinant hE1a has been produced according to both kinetic and spectroscopic criteria in our toolbox leading to the following conclusions: (i) The hE1a has recruited the dihydrolipoyl succinyltransferase (hE2o) and the dihydrolipoyl Dehydrogenase (hE3) components of the tricarboxylic acid cycle 2-Oxoglutarate Dehydrogenase Complex (OGDHc) for its activity. (ii) 2-Oxoglutarate (OG) and 2-oxoadipate (OA) could be oxidized by hE1a, however, hE1a displays an approximately 49-fold preference in catalytic efficiency for OA over OG, indicating that hE1a is specific to the 2-oxoadipate Dehydrogenase Complex. (iii) The hE1a forms the ThDP-enamine radical from OA according to electron paramagnetic resonance detection in the oxidative half reaction, and could produce superoxide and H2O2 from decarboxylation of OA in the forward physiological direction, as also seen with the 2-Oxoglutarate Dehydrogenase hE1o component. (iv) Once assembled to Complex with the same hE2o and hE3 components, the hE1o and hE1a display strikingly different regulation: both succinyl-CoA and glutaryl-CoA significantly reduced the hE1o activity, but not the activity of hE1a.
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the human krebs cycle 2 Oxoglutarate Dehydrogenase Complex creates an additional source of superoxide hydrogen peroxide from 2 oxoadipate as alternative substrate
Free Radical Biology and Medicine, 2017Co-Authors: Natalia S Nemeria, Pradeep Nareddy, Elena Guevara, Gary J Gerfen, Michal Szostak, Frank JordanAbstract:Recently, we reported that the human 2-Oxoglutarate Dehydrogenase (hE1o) component of the 2-Oxoglutarate Dehydrogenase Complex (OGDHc) could produce the reactive oxygen species superoxide and hydrogen peroxide (detected by chemical means) from its substrate 2-Oxoglutarate (OG), most likely concurrently with one-electron oxidation by dioxygen of the thiamin diphosphate (ThDP)-derived enamine intermediate to a C2α-centered radical (detected by Electron Paramagnetic Resonance) [Nemeria et al., 2014 [17]; Ambrus et al. 2015 [18]]. We here report that hE1o can also utilize the next higher homologue of OG, 2-oxoadipate (OA) as a substrate according to multiple criteria in our toolbox: (i) Both E1o-specific and overall Complex activities (NADH production) were detected using OA as a substrate; (ii) Two post-decarboxylation intermediates were formed by hE1o from OA, the ThDP-enamine and the C2α-hydroxyalkyl-ThDP, with nearly identical rates for OG and OA; (iii) Both OG and OA could reductively acylate lipoyl domain created from dihydrolipoyl succinyltransferase (E2o); (iv) Both OG and OA gave α-ketol carboligaton products with glyoxylate, but with opposite chirality; a finding that could be of utility in chiral synthesis; (v) Dioxygen could oxidize the ThDP-derived enamine from both OG and OA, leading to ThDP-enamine radical and generation of superoxide and H2O2. While the observed oxidation-reduction with dioxygen is only a side reaction of the predominant physiological product glutaryl-CoA, the efficiency of superoxide/ H2O2 production was 7-times larger from OA than from OG, making the reaction of OGDHc with OA one of the important superoxide/ H2O2 producers among 2-oxo acid Dehydrogenase Complexes in mitochondria.
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human 2 Oxoglutarate Dehydrogenase Complex e1 component forms a thiamin derived radical by aerobic oxidation of the enamine intermediate
Journal of Biological Chemistry, 2014Co-Authors: Natalia S Nemeria, Hetalben Patel, Laszlo Tretter, Attila Ambrus, Jieyu Zhou, Gary J Gerfen, Vera Adamvizi, Junjie Wang, Frank JordanAbstract:Abstract Herein are reported unique properties of the human 2-Oxoglutarate Dehydrogenase multienzyme Complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-Oxoglutarate Dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-Oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall Complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.
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assignment of function to histidines 260 and 298 by engineering the e1 component of the escherichia coli 2 Oxoglutarate Dehydrogenase Complex substitutions that lead to acceptance of substrates lacking the 5 carboxyl group
Biochemistry, 2011Co-Authors: Da Jeong Shim, Hetalben Patel, Natalia S Nemeria, Frank Jordan, Junjie Wang, Anand Balakrishnan, Jaeyoung Song, Edgardo T FarinasAbstract:The first component (E1o) of the Escherichia coli 2-Oxoglutarate Dehydrogenase Complex (OGDHc) was engineered to accept substrates lacking the 5-carboxylate group by subjecting H260 and H298 to saturation mutagenesis. Apparently, H260 is required for substrate recognition, but H298 could be replaced with hydrophobic residues of similar molecular volume. To interrogate whether the second component would allow synthesis of acyl-coenzyme A derivatives, hybrid Complexes consisting of recombinant components of OGDHc (o) and pyruvate Dehydrogenase (p) enzymes were constructed, suggesting that a different component is the “gatekeeper” for specificity for these two multienzyme Complexes in bacteria, E1p for pyruvate but E2o for 2-Oxoglutarate.
Kichiko Koike - One of the best experts on this subject based on the ideXlab platform.
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crystallization and preliminary x ray analysis of the full size cubic core of pig 2 Oxoglutarate Dehydrogenase Complex
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: K Suzuki, Kichiko Koike, Masahiko Koike, Takeshi Sekiguchi, Wataru Adachi, Noriyo Yamada, M Tsunoda, Akio TakenakaAbstract:The full-length (untruncated) dihydrolipoamide succinyltransferase from pig heart was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction patterns indicate that the crystal belongs to space group I432, with unit-cell parameter a = 189.9 A. The crystal structure has been preliminarily solved at 7 A resolution by the molecular-replacement method. The unit cell contains two cubic cores, in each of which 24 subunits of E2 are associated according to crystallographic 432 symmetry. At the corners of each cubic core, the catalytic domains of E2s form a trimer through tight interactions around the crystallographic threefold axes. In the electron-density maps, many small broad peaks are observed in regions expected to contain the remaining N-terminal domains (the E1/E3-binding domain and the lipoyl domain), suggesting flexibility of these domains relative to the core. The architecture of the cubic core is similar to that of the other truncated E2s. In the unit cell, however, the core–core contact occurs in a different direction from that found for the truncated proteins.
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cloning overexpression and mutagenesis of cdna encoding dihydrolipoamide succinyltransferase component of the porcine 2 Oxoglutarate Dehydrogenase Complex
FEBS Journal, 2000Co-Authors: Kichiko Koike, Takashi Suematsu, Masahiko EharaAbstract:Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2-Oxoglutarate Dehydrogenase (OGDH) Complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino-acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1–387), C- and N-terminal truncated PE2os, and site-directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET-11d and purified. The succinyltransferase activity of the rPE2o was about 2.2-fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube-like structure very similar to that of the native PE2o. Deletion of five amino-acid residues from the C-terminus resulted in a complete loss of both enzymatic activity and formation of the cube-like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C-terminal leucine triplet (Leu383–384–385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383–384–385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5-fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl-CoA. However, self-assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383–384–385 triplet are important residues in both the self-assembly and catalytic mechanism of PE2o.
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cloning and nucleotide sequence of the cdna encoding human 2 Oxoglutarate Dehydrogenase lipoamide
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Kichiko Koike, Yoshishige Urata, Shinji GotoAbstract:Abstract 2-Oxoglutarate Dehydrogenase (lipoamide) (( OGDH: 2-Oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-Oxoglutarate Dehydrogenase Complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.
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cloning and nucleotide sequence of the cdna encoding human 2 Oxoglutarate Dehydrogenase lipoamide
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Kichiko Koike, Yoshishige Urata, Shinji GotoAbstract:Abstract 2-Oxoglutarate Dehydrogenase (lipoamide) (( OGDH: 2-Oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-Oxoglutarate Dehydrogenase Complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.
Shinji Goto - One of the best experts on this subject based on the ideXlab platform.
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cloning and nucleotide sequence of the cdna encoding human 2 Oxoglutarate Dehydrogenase lipoamide
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Kichiko Koike, Yoshishige Urata, Shinji GotoAbstract:Abstract 2-Oxoglutarate Dehydrogenase (lipoamide) (( OGDH: 2-Oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-Oxoglutarate Dehydrogenase Complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.
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cloning and nucleotide sequence of the cdna encoding human 2 Oxoglutarate Dehydrogenase lipoamide
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: Kichiko Koike, Yoshishige Urata, Shinji GotoAbstract:Abstract 2-Oxoglutarate Dehydrogenase (lipoamide) (( OGDH: 2-Oxoglutarate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2 )) is a component enzyme of the 2-Oxoglutarate Dehydrogenase Complex. We have cloned a human cDNA encoding OGDH from a fetal liver cDNA library by plaque hybridization with a mixture of oligonucleotide probes designed from the amino acid sequences of porcine OGDH. This cDNA spans 4156 bases and contains an open reading frame of 3009 nucleotides encoding a presequence of 40 amino acid residues and a mature protein of 963 amino acid residues (Mr = 108,642). The size of the mRNA is approximately 4.2 kilobases. Comparison of the deduced amino acid sequence of the human OGDH with experimentally determined segments of porcine OGDH comprising 308 amino acid residues shows 93% sequence identity. The human OGDH has 37% sequence identity with 933 amino acid residues of the Escherichia coli OGDH and 40% sequence identity with 1014 residues of the yeast OGDH.
