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Mulchand S Patel - One of the best experts on this subject based on the ideXlab platform.
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How Dihydrolipoamide Dehydrogenase-binding protein binds Dihydrolipoamide Dehydrogenase in the human pyruvate Dehydrogenase complex.
Journal of Biological Chemistry, 2005Co-Authors: Ewa Ciszak, Lioubov G. Korotchkina, Anna Makal, Young Soo Hong, Ananthalakshmy K. Vettaikkorumakankauv, Mulchand S PatelAbstract:Abstract The Dihydrolipoamide Dehydrogenase-binding protein (E3BP) and the Dihydrolipoamide acetyltransferase (E2) component enzyme form the structural core of the human pyruvate Dehydrogenase complex by providing the binding sites for two other component proteins, Dihydrolipoamide Dehydrogenase (E3) and pyruvate Dehydrogenase (E1), as well as pyruvate Dehydrogenase kinases and phosphatases. Despite a high similarity between the primary structures of E3BP and E2, the E3-binding domain of human E3BP is highly specific to human E3, whereas the E1-binding domain of human E2 is highly specific to human E1. In this study, we characterized binding of human E3 to the E3-binding domain of E3BP by x-ray crystallography at 2.6-A resolution, and we used this structural information to interpret the specificity for selective binding. Two subunits of E3 form a single recognition site for the E3-binding domain of E3BP through their hydrophobic interface. The hydrophobic residues Pro133, Pro154, and Ile157 in the E3-binding domain of E3BP insert themselves into the surface of both E3 polypeptide chains. Numerous ionic and hydrogen bonds between the residues of three interacting polypeptide chains adjacent to the central hydrophobic patch add to the stability of the subcomplex. The specificity of pairing for human E3BP with E3 is interpreted from its subcomplex structure to be most likely due to conformational rigidity of the binding fragment of the E3-binding domain of E3BP and its exquisite amino acid match with the E3 target interface.
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mice deficient in Dihydrolipoamide Dehydrogenase show increased vulnerability to mptp malonate and 3 nitropropionic acid neurotoxicity
Journal of Neurochemistry, 2004Co-Authors: Peter Klivenyi, Mulchand S Patel, Anatoly A Starkov, Noel Y Calingasan, Gabrielle Gardian, Susan E Browne, Lichuan Yang, Parvesh Bubber, Gary E Gibson, Flint M BealAbstract:Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes α-ketoglutarate Dehydrogenase complex (KGDHC) and pyruvate Dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide Dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in Dihydrolipoamide Dehydrogenase (Dld+/–) show increased vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), malonate and 3-nitropropionic acid (3-NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase-positive neurons in the substantia nigra of Dld+/– mice than that seen in wild-type littermate controls. Striatal lesion volumes produced by malonate and 3-NP were significantly increased in Dld+/– mice. Studies of isolated brain mitochondria treated with 3-NP showed that both succinate-supported respiration and membrane potential were suppressed to a greater extent in Dld+/– mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.
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identification of a common mutation gly194cys in both arab moslem and ashkenazi jewish patients with Dihydrolipoamide Dehydrogenase e3 deficiency possible beneficial effect of vitamin therapy
Journal of Inherited Metabolic Disease, 2003Co-Authors: Y S Hong, Stanley H Korman, J Lee, P Ghoshal, V Barash, S Kang, M Kwon, Alisa Gutman, A Rachmel, Mulchand S PatelAbstract:Summary: Dihydrolipoamide Dehydrogenase (E3) deficiency with a clinical phenotype and genotype (Gly194Cys homozygous), previously identified only in Ashkenazi Jewish patients, was diagnosed in two Palestinian Arab siblings and two unrelated Ashkenazi Jewish patients. While three of the four patients died in childhood without specific treatment, the surviving patient at age 18 years may have benefited from long-term daily supplementation with a cocktail of riboflavin, biotin, coenzyme Q and carnitine.
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SITE-DIRECTED MUTAGENESIS OF HUMAN Dihydrolipoamide Dehydrogenase : ROLE OF LYSINE-54 AND GLUTAMATE-192 IN STABILIZING THE THIOLATE-FAD INTERMEDIATE
Protein Expression and Purification, 1999Co-Authors: Te-chung Liu, Lioubov G. Korotchkina, Nataraj N. Vettakkorumakankav, Young Soo Hong, Mulchand S PatelAbstract:The roles of lysine-54 (K54) and glutamate-192 (E192) of human Dihydrolipoamide Dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (
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targeted disruption of the murine Dihydrolipoamide Dehydrogenase gene dld results in perigastrulation lethality
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Mark T Johnson, Hsinsheng Yang, Terry Magnuson, Mulchand S PatelAbstract:The Dld gene product, known as Dihydrolipoamide Dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate Dehydrogenase, α-ketoglutarate Dehydrogenase, branched-chain α-ketoacid Dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld−/− class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld−/− embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.
Sisinthy Shivaji - One of the best experts on this subject based on the ideXlab platform.
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Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenase in Spermatozoa CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION, AND ACTIVITY DURING SPERM CAPACITATION
Journal of Biological Chemistry, 2005Co-Authors: Kasturi Mitra, Nandini Rangaraj, Sisinthy ShivajiAbstract:Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that Dihydrolipoamide Dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, Dihydrolipoamide Dehydrogenase along with its host complex, the pyruvate Dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of Dihydrolipoamide Dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of Dihydrolipoamide Dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, Dihydrolipoamide Dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.
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Novel Tyrosine-Phosphorylated Post-Pyruvate Metabolic Enzyme, Dihydrolipoamide Dehydrogenase, Involved in Capacitation of Hamster Spermatozoa
Biology of Reproduction, 2004Co-Authors: Kasturi Mitra, Sisinthy ShivajiAbstract:Capacitation is a process that confers fertilizing ability to spermatozoa and this critical event occurs in the development of mammalian spermatozoa during their transit through the female reproductive tract and precedes fertilization. Because spermatozoa are relatively silent in transcription and translation, posttranslational modifications perform the regulatory functions in these cells during capacitation. In this report, we identify a candidate protein, Dihydrolipoamide Dehydrogenase, which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing Dihydrolipoamide Dehydrogenase as a phosphoprotein. The cDNA sequence of hamster testes Dihydrolipoamide Dehydrogenase does not show any variation from the already reported mammalian Dihydrolipoamide Dehydrogenases. Downregulation of the activity of the hamster spermatozoal enzyme by its specific inhibitor, 5-methoxyindole-2-carboxylic acid, blocks acrosome reaction completely and hyperactivation partially, confirming the role of Dihydrolipoamide Dehydrogenase in hamster spermatozoal capacitation. We also delineate the temporal involvement of glucose and pyruvate-lactate, showing that the former is required in the earlier stages and the latter for the later stages of hamster spermatozoal capacitation. The essentiality of pyruvate-lactate during hyperactivation and acrosome reaction necessitates the involvement of the post-pyruvate-lactate enzyme, Dihydrolipoamide Dehydrogenase.
David W. Hough - One of the best experts on this subject based on the ideXlab platform.
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SITE-DIRECTED MUTAGENESIS AND HALOPHILICITY OF Dihydrolipoamide Dehydrogenase FROM THE HALOPHILIC ARCHAEON, HALOFERAX VOLCANII
European Journal of Biochemistry, 1997Co-Authors: Keith A. Jolley, Rupert J. M. Russell, David W. HoughAbstract:A homology-modelled structure of Dihydrolipoamide Dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens. Analysis of the halophilic enzyme structure identified a potential K+-binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non-halophilic Dihydrolipoamide Dehydrogenases, E423 is only present in the halophilic enzyme. Four site-directed mutations of the Haloferax Dihydrolipoamide Dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised. From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.
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Dihydrolipoamide Dehydrogenase from the halophilic archaeon Haloferax volcanii: homologous overexpression of the cloned gene.
Journal of bacteriology, 1996Co-Authors: Keith A. Jolley, David W. Hough, E Rapaport, W G Woods, M L Dyall-smithAbstract:The gene encoding Dihydrolipoamide Dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been subcloned and overexpressed in the parent organism by using the halophilic archaeal rRNA promoter. The recombinant protein has been purified to homogeneity and characterized with respect to its kinetic, molecular, and salt-dependent properties. A Dihydrolipoamide Dehydrogenase-minus mutant of H. volcanii has been created by homologous recombination with the subcloned gene after insertion of the mevinolin resistance determinant into the protein-coding region. To explore the physiological function of the Dihydrolipoamide Dehydrogenase, the growth properties of the mutant halophile have been examined.
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Dihydrolipoamide Dehydrogenase in the trypanosoma subgenus, trypanozoon.
Molecular and Biochemical Parasitology, 1994Co-Authors: Anthony J. Else, James F. Clarke, Anthony Willis, Simon A. Jackman, David W. HoughAbstract:The enzyme Dihydrolipoamide Dehydrogenase has been discovered and characterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, and Trypanosoma evansi. The three T. brucei species, which have insect procyclic forms biochemically distinct from their mammalian bloodstream forms, express Dihydrolipoamide Dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis constants for the enzyme from each of the three T. brucei species did not reveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against Dihydrolipoamide Dehydrogenase from the stereorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the Dihydrolipoamide Dehydrogenase from all three T. brucei species; by this method, the relative molecular masses of their Dihydrolipoamide Dehydrogenases are indistinguishable. Dihydrolipoamide Dehydrogenase was purified from both the bloodstream and the procyclic forms of T. b. brucei, and the N-terminal have been sequenced. These sequences are identical to the derived protein sequence of the cloned gene (Else et al., Eur. J. Biochem. 212 (1993) 423-429), but have a nine amino acid N-terminal truncation, giving an N-terminus equivalent to that of T. cruzi Dihydrolipoamide Dehydrogenase. The T. b. brucei Dihydrolipoamide Dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but with reduced specificity.
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Dihydrolipoamide Dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.
Biochemistry and Cell Biology, 1992Co-Authors: Nataraj Vettakkorumakankav, Kenneth J. Stevenson, David W. Hough, Matthew Davison, Janice YoungAbstract:Dihydrolipoamide Dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, Dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.
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Lipoic Acid and Dihydrolipoamide Dehydrogenase in Halophilic Archaeobacteria
General and Applied Aspects of Halophilic Microorganisms, 1991Co-Authors: Michael J. Danson, David W. Hough, Nataraj Vettakkorumakankav, Kenneth J. StevensonAbstract:We have discovered the presence of Dihydrolipoamide Dehydrogenase (DHLipDH) in the halophilic archaeobacteria, despite the fact that these organisms lack the multienzyme complexes with which this enzyme is associated in eubacteria and eucaryotes. We will discuss (a) the discovery, purification and characterisation of the halophilic DHLipDH, (b) the detection of the substrate (reduced lipoic acid) in halophilic archaeobacteria, (c) the discovery of DHLipDH and lipoic acid in several other archaeobacterial genera, and (d) the function of DHLipDH in the archaeobacteria and its evolution with respect to the multienzyme complexes of eubacteria and eucaryotes.
Kirill M. Popov - One of the best experts on this subject based on the ideXlab platform.
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Dihydrolipoamide Dehydrogenase-binding protein of the human pyruvate Dehydrogenase complex. DNA-derived amino acid sequence, expression, and reconstitution of the pyruvate Dehydrogenase complex
Journal of Biological Chemistry, 1997Co-Authors: Robert A. Harris, Melissa M. Bowker-kinley, Jingjau Jeng, Kirill M. PopovAbstract:Next Section Abstract Protein X, recently renamed Dihydrolipoamide Dehydrogenase-binding protein (E3BP), is required for anchoring Dihydrolipoamide Dehydrogenase (E3) to the Dihydrolipoamide transacetylase (E2) core of the pyruvate Dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E3BP of the human pyruvate Dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E2 component of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E2, 38% identity in its central region with the putative peripheral subunit-binding domain of E2, and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E2. The similarity in the latter domain stands in contrast to E3BP ofSaccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all Dihydrolipoamide acyltransferases is replaced by a serine residue in human E3BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E3BP and E2 resulted in the formation of an E2·E3BP subcomplex that spontaneously reconstituted the pyruvate Dehydrogenase complex in the presence of native E3 and recombinant pyruvate decarboxylase (E1).
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Dihydrolipoamide Dehydrogenase-binding protein of the human pyruvate Dehydrogenase complex. DNA-derived amino acid sequence, expression, and reconstitution of the pyruvate Dehydrogenase complex
The Journal of biological chemistry, 1997Co-Authors: Robert A. Harris, Melissa M. Bowker-kinley, Jingjau Jeng, Kirill M. PopovAbstract:Protein X, recently renamed Dihydrolipoamide Dehydrogenase-binding protein (E3BP), is required for anchoring Dihydrolipoamide Dehydrogenase (E3) to the Dihydrolipoamide transacetylase (E2) core of the pyruvate Dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E3BP of the human pyruvate Dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E2 component of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E2, 38% identity in its central region with the putative peripheral subunit-binding domain of E2, and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E2. The similarity in the latter domain stands in contrast to E3BP ofSaccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all Dihydrolipoamide acyltransferases is replaced by a serine residue in human E3BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E3BP and E2 resulted in the formation of an E2·E3BP subcomplex that spontaneously reconstituted the pyruvate Dehydrogenase complex in the presence of native E3 and recombinant pyruvate decarboxylase (E1).
Kasturi Mitra - One of the best experts on this subject based on the ideXlab platform.
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Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenase in Spermatozoa CORRELATION OF ITS LOCALIZATION, TYROSINE PHOSPHORYLATION, AND ACTIVITY DURING SPERM CAPACITATION
Journal of Biological Chemistry, 2005Co-Authors: Kasturi Mitra, Nandini Rangaraj, Sisinthy ShivajiAbstract:Spermatozoa are cells distinctly different from other somatic cells of the body, capacitation being one of the unique phenomena manifested by this gamete. We have shown earlier that Dihydrolipoamide Dehydrogenase, a post-pyruvate metabolic enzyme, undergoes capacitation-dependent tyrosine phosphorylation, and the functioning of the enzyme is required for hyperactivation (enhanced motility) and acrosome reaction of hamster spermatozoa (Mitra, K., and Shivaji, S. (2004) Biol. Reprod. 70, 887-899). In this report we have investigated the localization of this mitochondrial enzyme in spermatozoa revealing non-canonical extra-mitochondrial localization of the enzyme in mammalian spermatozoa. In hamster spermatozoa, Dihydrolipoamide Dehydrogenase along with its host complex, the pyruvate Dehydrogenase complex, are localized in the acrosome and in the principal piece of the sperm flagella. The localization of Dihydrolipoamide Dehydrogenase, however, appears to be in the mitochondria in the spermatocytes, but in spermatids it appears to show a juxtanuclear localization (like Golgi). The capacitation-dependent time course of tyrosine phosphorylation of Dihydrolipoamide Dehydrogenase appears to be different in the principal piece of the flagella and the acrosome in hamster spermatozoa. Activity assays of this bi-directional enzyme suggest a strong correlation between the tyrosine phosphorylation and the bi-directional enzyme activity. This is the first report of a direct correlation of the localization, tyrosine phosphorylation, and activity of the important metabolic enzyme, Dihydrolipoamide Dehydrogenase, implicating dual involvement and regulation of the enzyme during sperm capacitation.
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Novel Tyrosine-Phosphorylated Post-Pyruvate Metabolic Enzyme, Dihydrolipoamide Dehydrogenase, Involved in Capacitation of Hamster Spermatozoa
Biology of Reproduction, 2004Co-Authors: Kasturi Mitra, Sisinthy ShivajiAbstract:Capacitation is a process that confers fertilizing ability to spermatozoa and this critical event occurs in the development of mammalian spermatozoa during their transit through the female reproductive tract and precedes fertilization. Because spermatozoa are relatively silent in transcription and translation, posttranslational modifications perform the regulatory functions in these cells during capacitation. In this report, we identify a candidate protein, Dihydrolipoamide Dehydrogenase, which is a post-pyruvate metabolic enzyme, exhibiting tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing Dihydrolipoamide Dehydrogenase as a phosphoprotein. The cDNA sequence of hamster testes Dihydrolipoamide Dehydrogenase does not show any variation from the already reported mammalian Dihydrolipoamide Dehydrogenases. Downregulation of the activity of the hamster spermatozoal enzyme by its specific inhibitor, 5-methoxyindole-2-carboxylic acid, blocks acrosome reaction completely and hyperactivation partially, confirming the role of Dihydrolipoamide Dehydrogenase in hamster spermatozoal capacitation. We also delineate the temporal involvement of glucose and pyruvate-lactate, showing that the former is required in the earlier stages and the latter for the later stages of hamster spermatozoal capacitation. The essentiality of pyruvate-lactate during hyperactivation and acrosome reaction necessitates the involvement of the post-pyruvate-lactate enzyme, Dihydrolipoamide Dehydrogenase.