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Jan R. Andreesen - One of the best experts on this subject based on the ideXlab platform.
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thioredoxin elicits a new Dihydrolipoamide dehydrogenase activity by interaction with the electron transferring flavoprotein in clostridium litoralis and eubacterium acidaminophilum
Journal of Bacteriology, 1991Co-Authors: M. Meyer, D Dietrichs, Bernhard Schmidt, Jan R. AndreesenAbstract:Abstract The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing Dihydrolipoamide. The first one was found to be a genuine Dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by Dihydrolipoamide was the strongly preferred reaction. A second Dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the Dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low Dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.
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Peripheral localization of the Dihydrolipoamide dehydrogenase in the purinolytic anaerobe Clostridium cylindrosporum
Archives of Microbiology, 1991Co-Authors: D Dietrichs, M. Bahnweg, Frank Mayer, Jan R. AndreesenAbstract:Immunocytochemical localization experiments were performed with antibodies raised against the Dihydrolipoamide dehydrogenase protein (P3) of the glycine decarboxylase complex from clostridium cylindrosporum using the low-temperature procedure and protein A-gold technique. An association with the cytoplasmic membrane was indicated to about 65 (±10) % when cells were analyzed from the logarithmic growth phase. The unusual peripheral localization is discussed.
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Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of Dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria.
Journal of Bacteriology, 1990Co-Authors: D Dietrichs, Bernhard Schmidt, M. Meyer, Jan R. AndreesenAbstract:Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the Dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spectra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme of E. acidaminophilum, which exhibited by itself a Dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346-1354, 1989), the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no Dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the Dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the Dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring flavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the Dihydrolipoamide dehydrogenases purified from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to Dihydrolipoamide dehydrogenases known from other organisms.
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purification and comparative studies of Dihydrolipoamide dehydrogenases from the anaerobic glycine utilizing bacteria peptostreptococcus glycinophilus clostridium cylindrosporum and clostridium sporogenes
Journal of Bacteriology, 1990Co-Authors: D Dietrichs, Jan R. AndreesenAbstract:Three different Dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of Dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The Dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the Dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested. Images
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 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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Glycine decarboxylase and pyruvate dehydrogenase complexes share the same Dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis.
The Biochemical journal, 1996Co-Authors: Jacques Bourguignon, V Merand, S Rawsthorne, E Forest, Roland DouceAbstract:In order to compare the Dihydrolipoamide dehydrogenase associated with the pyruvate dehydrogenase complex (E3) with that associated with the glycine decarboxylase complex (L-protein), we report for the first time the purification and characterization of the E3 component from pea leaf mitochondria. The first 30 amino acids of the N-terminal sequence of the mature E3 protein are identical with those of the mature L-protein of the glycine decarboxylase complex. Electrospray ionization-mass spectrometric analysis of E3 and the L-protein gave exactly the same molecular mass of 49,753 +/- 5 Da. We have also confirmed the primary structure of the L-protein, in particular the C-terminal sequence, deduced from the cDNA published by Bourguignon, Macherel, Neuburger and Douce [(1992) Eur. J. Biochem. 204, 865-873]. Western-blot analysis shows that specific polyclonal antibodies raised against the L-protein recognize specifically both E3 and L-protein but not the porcine Dihydrolipoamide dehydrogenase. We conclude that, in pea leaf mitochondria, the pyruvate dehydrogenase and glycine decarboxylase complexes share the same Dihydrolipoamide dehydrogenase. We have also confirmed by MS analysis that the FAD is not covalently bound to the enzyme.
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isolation characterization and sequence analysis of a cdna clone encoding l protein the Dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea leaf mitochondria
FEBS Journal, 1992Co-Authors: Jacques Bourguignon, Michel Neuburger, David Macherel, Roland DouceAbstract:L-protein is the Dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a λ gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast Dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique Dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60 000 in different elution peaks where Dihydrolipoamide dehydrogenase activity has been detected. Second, Northernblot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the Dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
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Isolation, characterization, and sequence analysis of a cDNA clone encoding L‐protein, the Dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea‐leaf mitochondria
European Journal of Biochemistry, 1992Co-Authors: Jacques Bourguignon, Michel Neuburger, David Macherel, Roland DouceAbstract:L-protein is the Dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a λ gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast Dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique Dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60 000 in different elution peaks where Dihydrolipoamide dehydrogenase activity has been detected. Second, Northernblot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the Dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
Lester J. Reed - One of the best experts on this subject based on the ideXlab platform.
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Nucleotide sequence for yeast Dihydrolipoamide dehydrogenase (flavoprotein/conservation of sequence)
2016Co-Authors: Karen S. Browning, David J. Uhlinger, Lester J. Reed, Hans Trachsel, Michael AltmannAbstract:Rabbit antiserum to the Dihydrolipoamide dehydrogenase (Dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) component of the pyruvate dehydrogenase complex from bakers' yeast was used to screen plaques produced by a Agtll yeast cDNA library. A 2.1-kilobase insert was isolated that also hybridized to a 17-base mixed oligonucleotide probe corresponding to the amino-terminal sequence of the yeast Dihydrolipoamide dehydrogenase. The cDNA has a coding sequence of 499 amino acids that corresponds to a 21-residue signal peptide and a 478-residue mature protein (Mr = 51,558). Computer analysis shows that yeast dihydrofipoamide dehydrogenase has about 41% amino acid identity with Esch- erichia coli Dihydrolipoamide dehydrogenase. Particularly striking is the conservation of sequence in the active site region of the Dihydrolipoamide dehydrogenases from E. coli, yeast, and pig heart.
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crystal structure of the truncated cubic core component of the escherichia coli 2 oxoglutarate dehydrogenase multienzyme complex
Journal of Molecular Biology, 1998Co-Authors: James E Knapp, Lester J. Reed, David T Mitchell, Mohammad A Yazdi, S R Ernst, Marvin L HackertAbstract:Abstract The Dihydrolipoamide succinyltransferase (E2o) component of the 2-oxoglutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry. The catalytic domain (CD) of the E2o component catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 A resolution using molecular replacement phases derived from the structure of the catalytic domain from the Azotobacter vinelandii Dihydrolipoamide acetyltransferase (E2pCD). The refined model of the E. coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (Rfree = 0.249) for 9696 reflections between 20.0 and 3.0 A resolution. Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in comparison to the less stable A. vinelandii E2pCD 24mer. Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two 3-fold related subunits. Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT. A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from its counterpart in CAT (Asp199). Binding of the substrates to E2oCD is proposed to induce a change in the conformation of Asp379, allowing this residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT. Computer models of the active site of E2o complexed with Dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket. The residues which form this pocket (Ser330, Ser333, and His348) are probably responsible for E2o’s substrate specificity.
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Stoichiometry of binding of mature and truncated forms of the Dihydrolipoamide dehydrogenase-binding protein to the Dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.
Biochemistry, 1996Co-Authors: Cheol Young Maeng, Mohammed A. Yazdi, Lester J. ReedAbstract:The Dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of t...
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Overexpression and mutagenesis of the catalytic domain of Dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.
Biochemistry, 1990Co-Authors: Xiaoda Niu, James K. Stoops, Lester J. ReedAbstract:The inner core domain (residues approximately 221-454) of the Dihydrolipoamide acetyltransferase component (E2P) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [1-14C]acetyl-CoA to Dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the active site of E2p from S. cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues approximately 181-220) to the catalytic domain. All Dihydrolipoamide acyltransferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, J. R. (1987) FEMS Microbiol. Lett. 44, 417-422]. Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p. However, the Asp-431----Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)
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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Long-term regulation and promoter analysis of mammalian pyruvate dehydrogenase complex
Alpha-Keto Acid Dehydrogenase Complexes, 1996Co-Authors: Mulchand S Patel, Sharon S. Naik, Mark T. JohnsonAbstract:The mammalian pyruvate dehydrogenase complex (PDC) plays a key role in the irreversible decarboxylation of pyruvate derived from glucose and amino acids to form acetyl-CoA in the mitochondria. Acetyl-CoA is then utilized for either energy production by the tricarboxylic acid cycle or energy storage by the lipogenic pathway. This enzyme complex contains multiple copies of three catalytic components: pyruvate dehydrogenase (E1), Dihydrolipoamide acetytransferase (E2) and Dihydrolipoamide dehydrogenase (E3), two regulatory components (E1-kinase, phospho-E1 phosphatase) and one non-catalytic protein X (also referred to as E3-binding protein) (for review see: Reed, 1974; Patel and Roche, 1990; Behal et al., 1993).
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Spectroscopic Studies of the Characterization of Recombinant Human Dihydrolipoamide Dehydrogenase and Its Site-directed Mutants
Journal of Biological Chemistry, 1995Co-Authors: Te-chung Liu, Lioubov G. Korotchkina, Susannah L. Hyatt, Nataraj N. Vettakkorumakankav, Mulchand S PatelAbstract:Abstract In this paper, we report the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human Dihydrolipoamide dehydrogenase in Escherichia coli and detailed spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme. One mutation (K37E) has been identified in a patient lacking Dihydrolipoamide dehydrogenase activity and has been reported previously (Liu, T.-C., Kim, H., Arizmendi, C., Kitano, A., and Patel, M. S.(1993) Proc. Natl. Acad. Sci. USA. 90, 5186-5190), while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457). Circular dichroic and fluorescence spectroscopic data illustrate the role of these amino acids in maintaining the structure and function of human Dihydrolipoamide dehydrogenase. While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant. The K37E mutant shows very little deviation from the wild-type enzyme.
D Dietrichs - One of the best experts on this subject based on the ideXlab platform.
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thioredoxin elicits a new Dihydrolipoamide dehydrogenase activity by interaction with the electron transferring flavoprotein in clostridium litoralis and eubacterium acidaminophilum
Journal of Bacteriology, 1991Co-Authors: M. Meyer, D Dietrichs, Bernhard Schmidt, Jan R. AndreesenAbstract:Abstract The glycine-utilizing bacterium Clostridium litoralis contained two enzyme systems for oxidizing Dihydrolipoamide. The first one was found to be a genuine Dihydrolipoamide dehydrogenase, present only in low amounts. This enzyme had the typical dimeric structure with a subunit molecular mass of about 53 kDa; however, it reacted with both NADP (Km 0.11 mM) and NAD (Km 0.5 mM). The reduction of pyridine nucleotides by Dihydrolipoamide was the strongly preferred reaction. A second Dihydrolipoamide-oxidizing enzyme system consisted of the interaction of two proteins, the previously described NADP(H)-dependent electron-transferring flavoprotein (D. Dietrichs, M. Meyer, B. Schmidt, and J. R. Andreesen, J. Bacteriol. 172:2088-2095, 1990) and a thioredoxin. This enzyme system was responsible for most of the Dihydrolipoamide dehydrogenase activity in cell extracts. The thioredoxin did not bind to DEAE, was heat stable, and had a molecular mass of about 15 kDa. N-terminal amino acid analysis of the first 38 amino acid residues resulted in 38% homology to Escherichia coli thioredoxin and about 76% homology to a corresponding protein isolated from the physiologically close related Eubacterium acidaminophilum. The protein of the latter organism had a molecular mass of about 14 kDa and stimulated the low Dihydrolipoamide dehydrogenase activity of the corresponding flavoprotein. By this interaction with NADPH-dependent flavoproteins, a new assay system for thioredoxin was established. A function of thioredoxin in glycine metabolism of some anaerobic bacteria is proposed.
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Peripheral localization of the Dihydrolipoamide dehydrogenase in the purinolytic anaerobe Clostridium cylindrosporum
Archives of Microbiology, 1991Co-Authors: D Dietrichs, M. Bahnweg, Frank Mayer, Jan R. AndreesenAbstract:Immunocytochemical localization experiments were performed with antibodies raised against the Dihydrolipoamide dehydrogenase protein (P3) of the glycine decarboxylase complex from clostridium cylindrosporum using the low-temperature procedure and protein A-gold technique. An association with the cytoplasmic membrane was indicated to about 65 (±10) % when cells were analyzed from the logarithmic growth phase. The unusual peripheral localization is discussed.
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Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of Dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria.
Journal of Bacteriology, 1990Co-Authors: D Dietrichs, Bernhard Schmidt, M. Meyer, Jan R. AndreesenAbstract:Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the Dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spectra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme of E. acidaminophilum, which exhibited by itself a Dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346-1354, 1989), the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no Dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the Dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the Dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring flavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the Dihydrolipoamide dehydrogenases purified from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to Dihydrolipoamide dehydrogenases known from other organisms.
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purification and comparative studies of Dihydrolipoamide dehydrogenases from the anaerobic glycine utilizing bacteria peptostreptococcus glycinophilus clostridium cylindrosporum and clostridium sporogenes
Journal of Bacteriology, 1990Co-Authors: D Dietrichs, Jan R. AndreesenAbstract:Three different Dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of Dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The Dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the Dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested. Images