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Jerker M Olsson - One of the best experts on this subject based on the ideXlab platform.
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regeneration of the antioxidant ubiquinol by Lipoamide dehydrogenase thioredoxin reductase and glutathione reductase
Biofactors, 2003Co-Authors: Tomas Nordman, Elias S.j. Arnér, Ling Xia, Linda Bjorkhembergman, Anastassios E Damdimopoulos, Ivan Nalvarte, Giannis Spyrou, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. Lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by Lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.
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reduction of ubiquinone by Lipoamide dehydrogenase an antioxidant regenerating pathway
FEBS Journal, 2001Co-Authors: Tomas Nordman, Ling Xia, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synth ...
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ubiquinone is reduced by Lipoamide dehydrogenase and this reaction is potently stimulated by zinc
FEBS Letters, 1999Co-Authors: Jerker M Olsson, Ling Xia, Lennart Eriksson, Mikael BjornstedtAbstract:Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that Lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 μM in the presence and 4 μM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.
Ling Xia - One of the best experts on this subject based on the ideXlab platform.
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regeneration of the antioxidant ubiquinol by Lipoamide dehydrogenase thioredoxin reductase and glutathione reductase
Biofactors, 2003Co-Authors: Tomas Nordman, Elias S.j. Arnér, Ling Xia, Linda Bjorkhembergman, Anastassios E Damdimopoulos, Ivan Nalvarte, Giannis Spyrou, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. Lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by Lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.
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reduction of ubiquinone by Lipoamide dehydrogenase an antioxidant regenerating pathway
FEBS Journal, 2001Co-Authors: Tomas Nordman, Ling Xia, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synth ...
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ubiquinone is reduced by Lipoamide dehydrogenase and this reaction is potently stimulated by zinc
FEBS Letters, 1999Co-Authors: Jerker M Olsson, Ling Xia, Lennart Eriksson, Mikael BjornstedtAbstract:Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that Lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 μM in the presence and 4 μM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.
Mikael Bjornstedt - One of the best experts on this subject based on the ideXlab platform.
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regeneration of the antioxidant ubiquinol by Lipoamide dehydrogenase thioredoxin reductase and glutathione reductase
Biofactors, 2003Co-Authors: Tomas Nordman, Elias S.j. Arnér, Ling Xia, Linda Bjorkhembergman, Anastassios E Damdimopoulos, Ivan Nalvarte, Giannis Spyrou, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. Lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by Lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.
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reduction of ubiquinone by Lipoamide dehydrogenase an antioxidant regenerating pathway
FEBS Journal, 2001Co-Authors: Tomas Nordman, Ling Xia, Lennart Eriksson, Mikael Bjornstedt, Jerker M OlssonAbstract:Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synth ...
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ubiquinone is reduced by Lipoamide dehydrogenase and this reaction is potently stimulated by zinc
FEBS Letters, 1999Co-Authors: Jerker M Olsson, Ling Xia, Lennart Eriksson, Mikael BjornstedtAbstract:Ubiquinol is an endogenously synthesized lipid-soluble antioxidant. Regeneration of ubiquinol from the oxidized form is essential to the maintenance of its antioxidant function. We demonstrated that Lipoamide dehydrogenase can reduce ubiquinone to ubiquinol. Zinc increased the rate of the NADPH-dependent reduction more than 10-fold. The concentration ubiquinone resulting in the half-maximal rate of reduction was approximately 5 μM in the presence and 4 μM in the absence of zinc. These data may explain how ubiquinone is reduced to the active antioxidant ubiquinol, which plays such an important role in protecting against oxidative stress and lipid peroxidation.
Roland Douce - One of the best experts on this subject based on the ideXlab platform.
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interaction between the Lipoamide containing h protein and the Lipoamide dehydrogenase l protein of the glycine decarboxylase multienzyme system 1 biochemical studies
FEBS Journal, 2000Co-Authors: Michel Neuburger, Jacques Bourguignon, Ange Polidori, Emmanuel Pietre, Magali Faure, Agnes Jourdain, Bernard Pucci, Roland DouceAbstract:: Lipoamide dehydrogenase or dihydroLipoamide dehydrogenase (EC 1.8.1. 4) is the E3-protein component of the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. It is also the L-protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free Lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2-oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H-protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2-carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H-protein during the course of the reaction catalysed by the L-protein. This provided a valuable new tool with which to study the catalytic properties of the Lipoamide dehydrogenase. The L-protein displayed a much higher affinity for the dihydrolipoyl H-protein than for free dihydroLipoamide. The oxidation of the dihydrolipoyl H-protein was not affected by the presence of structurally related analogues (apoH-protein or octanoylated H-protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P-protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H-protein, containing the Lipoamide moiety, displayed Km values for the L-protein close to that found for the H-protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P-protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H-protein. From our results we concluded that no structural interaction is required for the L-protein to catalyse the oxidation of the dihydrolipoyl H-protein. We discuss the possibility that one function of the H-protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the Lipoamide dehydrogenase.
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interaction between the Lipoamide containing h protein and the Lipoamide dehydrogenase l protein of the glycine decarboxylase multienzyme system 2 crystal structures of h and l proteins
FEBS Journal, 2000Co-Authors: Magali Faure, Jacques Bourguignon, Michel Neuburger, Robert Ober, Claudine Cohenaddad, Larry C. Sieker, David Macherel, R. Kahn, Roland DouceAbstract:The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa Lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (Lipoamide dehydrogenase or dihydroLipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydroLipoamide dehydrogenase is similar to other known dihydroLipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded Lipoamide arm was locked into a cavity of the protein, the reduced Lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced Lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M.,
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interaction between the Lipoamide containing h protein and the Lipoamide dehydrogenase l protein of the glycine decarboxylase multienzyme system 2 crystal structures of h and l proteins
FEBS Journal, 2000Co-Authors: Magali Faure, Jacques Bourguignon, Michel Neuburger, Robert Ober, Claudine Cohenaddad, Larry C. Sieker, David Macherel, R. Kahn, Roland DouceAbstract:The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa Lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (Lipoamide dehydrogenase or dihydroLipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydroLipoamide dehydrogenase is similar to other known dihydroLipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded Lipoamide arm was locked into a cavity of the protein, the reduced Lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced Lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Pietre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering experiments reported herein.
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refined structures at 2 and 2 2 a resolution of two forms of the h protein a Lipoamide containing protein of the glycine decarboxylase complex
Acta Crystallographica Section D-biological Crystallography, 1995Co-Authors: Serge Pares, Michel Neuburger, Claudine Cohenaddad, Larry C. Sieker, Roland DouceAbstract:H-protein, a 14 kDa lipoic acid-containing protein is a component of the glycine decarboxylase complex. This complex which consists of four protein components (P-, H-, T- and L-protein) catalyzes the oxidative decarboxylation of glycine. The mechanistic heart of the complex is provided by the lipoic acid attached to a lysine residue of the H-protein. It undergoes a cycle of transformations, i.e. reductive methylamination, methylamine transfer, and electron transfer. We present details of the crystal structures of the H-protein, in its two forms, H-ProOx with oxidized Lipoamide and H-ProMet with methylamine-loaded Lipoamide. X-ray diffraction data were collected from crystals of H-ProOx to 2 and H-ProMet to 2.2 A resolution. The final R-factor value for the H-ProOx is 18.5% for data with F > 2σ. in the range of 8.0–2.0 A resolution. The refinement confirmed our previous model, refined to 2.6 A, of a β-fold sandwich structure with two β-sheets. The Lipoamide arm attached to Lys63, located in the loop of a hairpin conformation, is clearly visible at the surface of the protein. The H-ProMet has been crystallized in orthorhombic and monoclinic forms and the structures were solved by molecular replacement, starting from the H-ProOx model. The orthorhombic structure has been refined with a final R-factor value of 18.5% for data with F > 2σ in the range of 8.0–2.2 A resolution. The structure of the monoclinic form has been refined with a final R-factor value of 17.5% for data with F > 2σ in the range of 15.0–3.0 A. In these two structures which have similar packing, the protein conformation is identical to the conformation found in the H-ProOx. The main change lies in the position of the Lipoamide group which has moved significantly when loaded with methylamine. In this case the methylamine-Lipoamide group is tucked into a cleft at the surface of the protein where it is stabilized by hydrogen bonds and hydrophobic contacts. Thus, it is totally protected and not free to move in aqueous solvent. In addition, the H-protein presents some sequence and structural analogies with other lipoate- and biotin-containing proteins and also with proteins of the phosphoenolpyruvate:sugar phosphotransferase system.
John S. Blanchard - One of the best experts on this subject based on the ideXlab platform.
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Catalysis of diaphorase reactions by Mycobacterium tuberculosis Lipoamide dehydrogenase occurs at the EH4 level.
Biochemistry, 2003Co-Authors: Argyrides Argyrou, Bruce A. Palfey, Guangxing Sun, John S. BlanchardAbstract:Lipoamide dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH(2)), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH(4)), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O(2)). To determine the mechanism of these "diaphorase" reactions, we generated the EH(2) and EH(4) forms of Mycobacterium tuberculosis Lipoamide dehydrogenase and rapidly mixed these enzyme forms with d,l-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O(2), in a stopped-flow spectrophotometer at pH 7.5 and 4 degrees C. EH(2) reduced d,l-lipoylpentanoate >/=100 times faster than EH(4) did. Conversely, EH(4) reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH(2), respectively. Comparison of the rates of reduction of the above substrates by EH(2) and EH(4) with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH(2) while reduction of diaphorase substrates occurs with EH(4).
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mycobacterium tuberculosis Lipoamide dehydrogenase is encoded by rv0462 and not by the lpda or lpdb genes
Biochemistry, 2001Co-Authors: Argyrides Argyrou, John S. BlanchardAbstract:The gene encoding dihydroLipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD+ in the absence of d,l-Lipoamide, demonstrated that the enzyme uses a ping−pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C4-proS position of the nicotinamide ring are small [D(V/K)NADH = 1.12 ± 0.15, DVapp = 1.05 ± 0.07] when d,l-Lipoamide is the oxidant but large and equivalent [D(V/K)NADH = DV = 2.95 ± 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with D(V/K)AP...
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catalytic properties of Lipoamide dehydrogenase from mycobacterium smegmatis
Archives of Biochemistry and Biophysics, 1997Co-Authors: Jovita Marcinkeviciene, John S. BlanchardAbstract:Abstract Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold. Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens Lipoamide dehydrogenases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530–550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfide–dithiol. The kinetic mechanism of the forward (NAD + reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD + and NADH. The rate of Lipoamide reduction was found to depend upon the NAD + /NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydroLipoamide, the enzyme displayed high oxidase activity (30% of the Lipoamide reduction rate), hydrogen and t -butyl peroxide reductase activity (10% of the Lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (∼200% of the Lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The Lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.