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Bernhard J Eikmanns - One of the best experts on this subject based on the ideXlab platform.
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genetic and functional analysis of the soluble Oxaloacetate decarboxylase from corynebacterium glutamicum
Journal of Bacteriology, 2010Co-Authors: Simon Klaffl, Bernhard J EikmannsAbstract:Soluble, divalent cation-dependent Oxaloacetate decarboxylases (ODx) catalyze the irreversible decarboxylation of Oxaloacetate to pyruvate and CO2. Although these enzymes have been characterized in different microorganisms, the genes that encode them have not been identified, and their functions have been only poorly analyzed so far. In this study, we purified a soluble ODx from wild-type C. glutamicum about 65-fold and used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis and peptide mass fingerprinting for identification of the corresponding odx gene. Inactivation and overexpression of odx led to an absence of ODx activity and to a 30-fold increase in ODx specific activity, respectively; these findings unequivocally confirmed that this gene encodes a soluble ODx. Transcriptional analysis of odx indicated that there is a leaderless transcript that is organized in an operon together with a putative S-adenosylmethionine-dependent methyltransferase gene. Biochemical analysis of ODx revealed that the molecular mass of the native enzyme is about 62 ± 1 kDa and that the enzyme is composed of two ∼29-kDa homodimeric subunits and has a Km for Oxaloacetate of 1.4 mM and a Vmax of 201 μmol of Oxaloacetate converted per min per mg of protein, resulting in a kcat of 104 s−1. Introduction of plasmid-borne odx into a pyruvate kinase-deficient C. glutamicum strain restored growth of this mutant on acetate, indicating that a high level of ODx activity redirects the carbon flux from Oxaloacetate to pyruvate in vivo. Consistently, overexpression of the odx gene in an l-lysine-producing strain of C. glutamicum led to accumulation of less l-lysine. However, inactivation of the odx gene did not improve l-lysine production under the conditions tested.
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the pep pyruvate Oxaloacetate node as the switch point for carbon flux distribution in bacteria
Fems Microbiology Reviews, 2005Co-Authors: Uwe Sauer, Bernhard J EikmannsAbstract:In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)–pyruvate–Oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (Oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP–pyruvate–Oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP–pyruvate–Oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP–pyruvate–Oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.
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The PEP–pyruvate–Oxaloacetate node as the switch point for carbon flux distribution in bacteria
Fems Microbiology Reviews, 2004Co-Authors: Uwe Sauer, Bernhard J EikmannsAbstract:In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)–pyruvate–Oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (Oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP–pyruvate–Oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP–pyruvate–Oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP–pyruvate–Oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.
Jose Castillo - One of the best experts on this subject based on the ideXlab platform.
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human recombinant glutamate Oxaloacetate transaminase 1 got1 supplemented with Oxaloacetate induces a protective effect after cerebral ischemia
Cell Death and Disease, 2014Co-Authors: Maria Perezmato, Tomas Sobrino, Pedro Ramoscabrer, Jose Castillo, Miguel Blanco, Angela Ruban, David Mirelman, Pablo Menendez, Francisco CamposAbstract:Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate Oxaloacetate transaminase 1 (GOT1) activation by means of Oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of Oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of Oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with Oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of Oxaloacetate seems to be a successful approach for stroke treatment
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Oxaloacetate a novel neuroprotective for acute ischemic stroke
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Francisco Campos, Tomas Sobrino, Pedro Ramoscabrer, Jose CastilloAbstract:Abstract It is well established that glutamate acts as an important mediator of neuronal degeneration during cerebral ischemia. Different kind of glutamate antagonists have been used to reduce the deleterious effects of glutamate. However, their preclinical success failed to translate into practical treatments. Far from the classical use of glutamate antagonists employed so far, the systemic administration of Oxaloacetate represents a novel neuroprotective strategy to minimize the deleterious effect of glutamate in the brain tissue after ischemic stroke. The neuroprotective effect of Oxaloacetate is based on the capacity of this molecule to reduce the brain and blood glutamate levels as a result of the activation of the blood-resident enzyme glutamate-Oxaloacetate transaminase. Here we review the recent experimental and clinical results where it is demonstrated the potential applicability of Oxaloacetate as a novel and powerful neuroprotective treatment against ischemic stroke.
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neuroprotection by glutamate Oxaloacetate transaminase in ischemic stroke an experimental study
Journal of Cerebral Blood Flow and Metabolism, 2011Co-Authors: Francisco Campos, Tomas Sobrino, Pedro Ramoscabrer, Maria Perezmato, Barbara Argibay, Jesus Agulla, Raquel Rodriguezgonzalez, David Brea, Jose CastilloAbstract:As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain–blood gradient. In this regard, the ability of glutamate Oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, Oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after Oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.
Peter Dimroth - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the carboxyltransferase domain of the Oxaloacetate decarboxylase na pump from vibrio cholerae
Journal of Molecular Biology, 2006Co-Authors: Remo Studer, Pius Dahinden, Weiwu Wang, Yolanda Auchli, Xiaodan Li, Peter DimrothAbstract:Abstract Oxaloacetate decarboxylase is a membrane-bound multiprotein complex that couples Oxaloacetate decarboxylation to sodium ion transport across the membrane. The initial reaction catalyzed by this enzyme machinery is the carboxyl transfer from Oxaloacetate to the prosthetic biotin group. The crystal structure of the carboxyltransferase at 1.7 A resolution shows a dimer of α 8 β 8 barrels with an active site metal ion, identified spectroscopically as Zn 2+ , at the bottom of a deep cleft. The enzyme is completely inactivated by specific mutagenesis of Asp17, His207 and His209, which serve as ligands for the Zn 2+ metal ion, or by Lys178 near the active site, suggesting that Zn 2+ as well as Lys178 are essential for the catalysis. In the present structure this lysine residue is hydrogen-bonded to Cys148. A potential role of Lys178 as initial acceptor of the carboxyl group from Oxaloacetate is discussed.
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aspartate 203 of the Oxaloacetate decarboxylase beta subunit catalyses both the chemical and vectorial reaction of the na pump
The EMBO Journal, 1996Co-Authors: M Di Berardino, Peter DimrothAbstract:Abstract We report here a new mode of coupling between the chemical and vectorial reaction explored for the Oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfer from Oxaloacetate. The two conserved aspartic acid residues within putative membrane-spanning domains of the beta-subunit (Asp149 and Asp203) were exchanged by site-directed mutagenesis. Mutants D149Q and D149E retained Oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid-labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by Oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of Oxaloacetate decarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.
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On the mechanism of sodium ion translocation by Oxaloacetate decarboxylase of Klebsiella pneumoniae
Biochemistry, 1993Co-Authors: Peter Dimroth, Anna ThomerAbstract:Proteoliposomes reconstituted with purified Oxaloacetate decarboxylase of Klebsiella pneumoniae catalyzed the uptake of Na + ions upon Oxaloacetate decarboxylation. The degree of coupling between the chemical and the vectorial reaction is dependent on the reconstitution conditions, and with the best preparations approaches a stoichiometry of two Na + ions per decarboxylation of one Oxaloacetate. This coupling ratio is observed only in the absence of a Δμ Na+ , immediately after Oxaloacetate addition. The ratio gradually declines during development of the electrochemical Na + ion gradient and becomes zero in the steady state
Francisco Campos - One of the best experts on this subject based on the ideXlab platform.
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human recombinant glutamate Oxaloacetate transaminase 1 got1 supplemented with Oxaloacetate induces a protective effect after cerebral ischemia
Cell Death and Disease, 2014Co-Authors: Maria Perezmato, Tomas Sobrino, Pedro Ramoscabrer, Jose Castillo, Miguel Blanco, Angela Ruban, David Mirelman, Pablo Menendez, Francisco CamposAbstract:Blood glutamate scavenging is a novel and attractive protecting strategy to reduce the excitotoxic effect of extracellular glutamate released during ischemic brain injury. Glutamate Oxaloacetate transaminase 1 (GOT1) activation by means of Oxaloacetate administration has been used to reduce the glutamate concentration in the blood. However, the protective effect of the administration of the recombinant GOT1 (rGOT1) enzyme has not been yet addressed in cerebral ischemia. The aim of this study was to analyze the protective effect of an effective dose of Oxaloacetate and the human rGOT1 alone and in combination with a non-effective dose of Oxaloacetate in an animal model of ischemic stroke. Sixty rats were subjected to a transient middle cerebral artery occlusion (MCAO). Infarct volumes were assessed by magnetic resonance imaging (MRI) before treatment administration, and 24 h and 7 days after MCAO. Brain glutamate levels were determined by in vivo MR spectroscopy (MRS) during artery occlusion (80 min) and reperfusion (180 min). GOT activity and serum glutamate concentration were analyzed during the occlusion and reperfusion period. Somatosensory test was performed at baseline and 7 days after MCAO. The three treatments tested induced a reduction in serum and brain glutamate levels, resulting in a reduction in infarct volume and sensorimotor deficit. Protective effect of rGOT1 supplemented with Oxaloacetate at 7 days persists even when treatment was delayed until at least 2 h after onset of ischemia. In conclusion, our findings indicate that the combination of human rGOT1 with low doses of Oxaloacetate seems to be a successful approach for stroke treatment
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Oxaloacetate a novel neuroprotective for acute ischemic stroke
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Francisco Campos, Tomas Sobrino, Pedro Ramoscabrer, Jose CastilloAbstract:Abstract It is well established that glutamate acts as an important mediator of neuronal degeneration during cerebral ischemia. Different kind of glutamate antagonists have been used to reduce the deleterious effects of glutamate. However, their preclinical success failed to translate into practical treatments. Far from the classical use of glutamate antagonists employed so far, the systemic administration of Oxaloacetate represents a novel neuroprotective strategy to minimize the deleterious effect of glutamate in the brain tissue after ischemic stroke. The neuroprotective effect of Oxaloacetate is based on the capacity of this molecule to reduce the brain and blood glutamate levels as a result of the activation of the blood-resident enzyme glutamate-Oxaloacetate transaminase. Here we review the recent experimental and clinical results where it is demonstrated the potential applicability of Oxaloacetate as a novel and powerful neuroprotective treatment against ischemic stroke.
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neuroprotection by glutamate Oxaloacetate transaminase in ischemic stroke an experimental study
Journal of Cerebral Blood Flow and Metabolism, 2011Co-Authors: Francisco Campos, Tomas Sobrino, Pedro Ramoscabrer, Maria Perezmato, Barbara Argibay, Jesus Agulla, Raquel Rodriguezgonzalez, David Brea, Jose CastilloAbstract:As ischemic stroke is associated with an excessive release of glutamate into the neuronal extracellular space, a decrease in blood glutamate levels could provide a mechanism to remove it from the brain tissue, by increasing the brain–blood gradient. In this regard, the ability of glutamate Oxaloacetate transaminase (GOT) to metabolize glutamate in blood could represent a potential neuroprotective tool for ischemic stroke. This study aimed to determine the neuroprotective effects of GOT in an animal model of cerebral ischemia by means of a middle cerebral arterial occlusion (MCAO) following the Stroke Therapy Academic Industry Roundtable (STAIR) group guidelines. In this animal model, Oxaloacetate-mediated GOT activation inhibited the increase of blood and cerebral glutamate after MCAO. This effect is reflected in a reduction of infarct size, smaller edema volume, and lower sensorimotor deficits with respect to controls. Magnetic resonance spectroscopy confirmed that the increase of glutamate levels in the brain parenchyma after MCAO is inhibited after Oxaloacetate-mediated GOT activation. These findings show the capacity of the GOT to remove glutamate from the brain by means of blood glutamate degradation, and suggest the applicability of this enzyme as an efficient and novel neuroprotective tool against ischemic stroke.
Alexander Zlotnik - One of the best experts on this subject based on the ideXlab platform.
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the effect of blood glutamate scavengers Oxaloacetate and pyruvate on neurological outcome in a rat model of subarachnoid hemorrhage
Neurotherapeutics, 2012Co-Authors: M. Boyko, Benjamin F. Gruenbaum, Shaun E. Gruenbaum, Israel Melamed, Sharon Ohayon, Akiva Leibowitz, Evgeny Brotfain, Yoash Shapira, Alexander ZlotnikAbstract:Blood glutamate scavengers have been shown to effectively reduce blood glutamate concentrations and improve neurological outcome after traumatic brain injury and stroke in rats. This study investigates the efficacy of blood glutamate scavengers Oxaloacetate and pyruvate in the treatment of subarachnoid hemorrhage (SAH) in rats. Isotonic saline, 250 mg/kg Oxaloacetate, or 125 mg/kg pyruvate was injected intravenously in 60 rats, 60 minutes after induction of SAH at a rate of 0.1 ml/100 g/min for 30 minutes. There were 20 additional rats that were used as a sham-operated group. Blood samples were collected at baseline and 90 minutes after SAH. Neurological performance was assessed at 24 h after SAH. In half of the rats, glutamate concentrations in the cerebrospinal fluid were measured 24 h after SAH. For the remaining half, the blood brain barrier permeability in the frontal and parieto-occipital lobes was measured 48 h after SAH. Blood glutamate levels were reduced in rats treated with Oxaloacetate or pyruvate at 90 minutes after SAH (p < 0.001). Cerebrospinal fluid glutamate was reduced in rats treated with pyruvate (p < 0.05). Neurological performance was significantly improved in rats treated with Oxaloacetate (p < 0.05) or pyruvate (p < 0.01). The breakdown of the blood brain barrier was reduced in the frontal lobe in rats treated with pyruvate (p < 0.05) and in the parieto-occipital lobes in rats treated with either pyruvate (p < 0.01) or Oxaloacetate (p < 0.01). This study demonstrates the effectiveness of blood glutamate scavengers Oxaloacetate and pyruvate as a therapeutic neuroprotective strategy in a rat model of SAH.
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the neuroprotective effects of Oxaloacetate in closed head injury in rats is mediated by its blood glutamate scavenging activity evidence from the use of maleate
Journal of Neurosurgical Anesthesiology, 2009Co-Authors: Alexander Zlotnik, Shaun E. Gruenbaum, Evgeny Brotfain, Yoash Shapira, Alan A Artru, Irene Rozet, Michael Dubilet, Sergey Tkachov, Yael Klin, Vivian I TeichbergAbstract:IntroductionTreatment with Oxaloacetate after traumatic brain injury has been shown to decrease blood glutamate levels and protect against the neurotoxic effects of glutamate on the brain. A number of potential mechanisms have been suggested to explain Oxaloacetate-induced neuroprotection. We hypoth
