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PubMed2001

Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway.

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 synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min(-1) in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP(+) ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.

Animals↗
PubMed1996

Lipoamide dehydrogenase activity in lymphocytes.

To assess the suitability of lymphocytes for patient diagnosis and carrier detection of lipoamide dehydrogenase deficiency, the activity of lipoamide dehydrogenase was determined in lymphocytes of six patients, seven obligate heterozygotes and 32 healthy controls. In healthy controls, lipoamide dehydrogenase activity was 80.7 +/- 23.6 nmol/min/mg protein, in obligate heterozygotes it was 36.0 +/- 12.1 nmol/min/mg protein and in the patients it was 13.3 +/- 5.1 nmol/min/mg protein. For the purpose of standardization, the results were also calculated as lipoamide dehydrogenase/citrate synthase activity ratio. The activity of lipoamide dehydrogenase and the lipoamide dehydrogenase/citrate synthase ratio differed significantly between the three groups (P < 0.005). We conclude that lymphocytes are suitable for the diagnosis of lipoamide dehydrogenase deficiency and for carrier detection.

Blood Preservation↗
PubMed1991

Lipoamide dehydrogenase of Staphylococcus aureus: nucleotide sequence and sequence analysis.

A complex of four proteins was previously isolated from Staphylococcus aureus. The complex had a strong interaction with membrane bound ribosomes, which suggested that it may be involved in protein secretion. However, the complex was identified as pyruvate dehydrogenase (PDH), which disproved the direct role of the complex in protein secretion. Here we report the nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, which encodes lipoamide dehydrogenase (LPD). The pdhD gene encodes a protein of 468 amino acids, with a molecular mass of 49.5 kDa. The protein is closely related to other lipoamide dehydrogenases from bacteria and eukaryotes. The possible role of membrane bound lipoamide dehydrogenase is briefly discussed.

Amino Acid Sequence↗
PubMed1989

Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched-chain-oxoacid dehydrogenase of Pseudomonas putida.

The production of two lipoamide dehydrogenases by Pseudomonas is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putida grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C. Codon usage is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The Mr of the protein encoded is 48,164 and when the Mr of FAD is added, the total Mr is 48,949, which is very close to the value of 49,000 obtained by SDS-polyacrylamide gel electrophoresis. Similarity comparisons of LPD-val with sequences of three other lipoamide dehydrogenases showed that LPD-val was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1984

Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart.

The effect of NAD+ on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH2) couple in the presence on NAD+ were -218 mV and -251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD+ binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD+. Fluorescence quenching of bound FAD by NAD+ was released by phenobarbital. The results suggest that NAD+ strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD+ acts not only as an acceptor of electrons from EH2, but also as an effector in the flavin-disulfide interaction of EH2.

Animals↗
PubMed1997

Hymenolepis diminuta: mitochondrial NADH --> NAD transhydrogenation and the lipoamide dehydrogenase system.

The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.

Amino Acid Sequence↗
PubMed1987

Cloning and characterization of the gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae.

The LPD1 gene of S. cerevisiae, which encodes lipoamide dehydrogenase (EC 1.8.1.4), has been cloned and characterized. The LPD1 gene is present as a single copy in the yeast genome and is transcribed to give a polyadenylated mRNA species of approximately 2.0 kb. The synthesis of lipoamide dehydrogenase in yeast is subject to carbon catabolite repression since both the level of the LPD1 transcript and the accumulation of the lipoamide dehydrogenase subunit polypeptide were greatly reduced in wild-type cells grown on glucose compared to those grown on a variety of non-fermentable carbon sources. Strains defective in LPD1 but transformed with the LPD1 gene on a high copy number vector exhibited elevated levels of the LPD1 transcript as well as increased lipoamide dehydrogenase activity when grown on glycerol. Immunoblotting experiments confirmed that such transformants over-expressed lipoamide dehydrogenase protein. Transcription from the LPD1 sequence on plasmid pGP1 still appeared to be subject to some catabolite repression despite the presence of multiple copies of the plasmid in the cell.

Autoradiography↗
PubMed1989

Isolation of a third lipoamide dehydrogenase from Pseudomonas putida.

Pseudomonads are the only organisms so far known to produce two lipoamide dehydrogenases (LPDs), LPD-Val and LPD-Glc. LPD-Val is the specific E3 component of branched-chain oxoacid dehydrogenase, and LPD-Glc is the E3 component of 2-ketoglutarate and possibly pyruvate dehydrogenases and the L-factor of the glycine oxidation system. Three mutants of Pseudomonas putida, JS348, JS350, and JS351, affected in lpdG, the gene encoding LPD-Glc, have been isolated; all lacked 2-ketoglutarate dehydrogenase, but two, JS348 and JS351, had normal pyruvate dehydrogenase activity. The pyruvate and 2-ketoglutarate dehydrogenases of the wild-type strain of P. putida were both inhibited by anti-LPD-Glc, but the pyruvate dehydrogenase of the lpdG mutants was not inhibited, suggesting that the mutant pyruvate dehydrogenase E3 component was different from that of the wild type. The lipoamide dehydrogenase present in one of the lpdG mutants, JS348, was isolated and characterized. This lipoamide dehydrogenase, provisionally named LPD-3, differed in molecular weight, amino acid composition, and N-terminal amino acid sequence from LPD-Glc and LPD-Val. LPD-3 was clearly a lipoamide dehydrogenase as opposed to a mercuric reductase or glutathione reductase. LPD-3 was about 60% as effective as LPD-Glc in restoring 2-ketoglutarate dehydrogenase activity and completely restored pyruvate dehydrogenase activity in JS350. These results suggest that LPD-3 is a lipoamide dehydrogenase associated with an unknown multienzyme complex which can replace LPD-Glc as the E3 component of pyruvate and 2-ketoglutarate dehydrogenases in lpdG mutants.

Amino Acid Sequence↗
PubMed1975

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.

Alkanes↗
PubMed1998

Lipoamide dehydrogenase deficiency: a newly discovered cause of acute hepatitis in adults.

Lipoamide dehydrogenase deficiency is a rare disease, manifested in early childhood by lactic acidemia, progressive neurological damage and death in most cases. We report a case of lipoamide dehydrogenase deficiency in a 34-year-old Ashkenazi-Jewish woman. The deficiency manifested as acute hepatitis without cognitive impairment or acidosis. The patient's brother also had lipoamide dehydrogenase deficiency, diagnosed at the age of 20, and manifested as hepatocellular damage, lactic acidemia and myoglobinuria. We assume that the trigger for this hepatocellular damage was prolonged fasting, and that otherwise the patient might have gone undiagnosed. Other cases in Ashkenazi Jews of mild lipoamide dehydrogenase deficiency with hepatocellular injury but without central nervous system involvement are reviewed.

Acute Disease↗
PubMed1999

Nitrofuran drugs as common subversive substrates of Trypanosoma cruzi lipoamide dehydrogenase and trypanothione reductase.

Lipoamide dehydrogenase (LipDH), trypanothione reductase (TR), and glutathione reductase (GR) catalyze the NAD(P)H-dependent reduction of disulfide substrates. TR occurs exclusively in trypanosomatids which lack a GR. Besides their physiological reactions, the flavoenzymes catalyze the single-electron reduction of nitrofurans with the concomitant generation of superoxide anions. Here, we report on the interaction of clinically used antimicrobial nitrofurans with LipDH and TR from Trypanosoma cruzi, the causative agent of Chagas' disease (South American trypanosomiasis), in comparison to mammalian LipDH and GR. The compounds were studied as inhibitors and as subversive substrates of the enzymes. None of the nitrofurans inhibited LipDH, although they did interfere with the disulfide reduction of TR and GR. When the compounds were studied as substrates, T. cruzi LipDH showed a high rate of nitrofuran reduction and was even more efficient than its mammalian counterpart. Several derivatives were also effective subversive substrates of TR, but the respective reaction with human GR was negligible. Nifuroxazide, nifuroxime, and nifurprazine proved to be the most promising derivatives since they were redox-cycled by both T. cruzi LipDH and TR and had pronounced antiparasitic effects in cultures of T. cruzi and Trypanosoma brucei. The results suggest that those nitrofuran derivatives which interact with both parasite flavoenzymes should be revisited as trypanocidal drugs.

Animals↗
PubMed1982

Intracellular distribution and biosynthesis of lipoamide dehydrogenase in rat liver.

Lipoamide dehydrogenase (LADase) was purified to homogeneity from rat liver mitochondria, and the intracellular distribution and biosynthesis of the LADase were investigated with antibody prepared against the purified enzyme. 1) LADase activity was mostly found in mitochondria; the activity in cytosol was about one-tenth of that in mitochondria. 2) LADase in the crude mitochondrial and cytosolic extracts and the purified LADase were immunologically identical as judged from the Ouchterlony double diffusion test. These LADases were indistinguishable from each other on immunochemical titration; i.e., the amount of LADase precipitated by a fixed amount of the anti-LADase antibody was the same for all the preparations. However, cytosolic LADase activity was inhibited by the antibody more strongly than mitochondrial LADase activity. 3) Two min after intravenous injection of [35S]methionine, more radioactivity was incorporated into cytosolic LADase than into the mitochondrial enzyme in the liver. This result suggests that localization of LADase in the cytosolic fraction is not an artifact due to leakage from mitochondria during homogenization of rat liver. 4) LADase was synthesized predominantly on free ribosomes, which indicates that LADase is synthesized on cytoplasmic ribosomes and translocated into mitochondria just as other mitochondrial proteins are. 5) After cell-free protein synthesis with post-mitochondrial supernatant, radioactivity immunoprecipitated with anti-LADase antibody was detected as a major peak with the same molecular weight as the purified LADase.

Animals↗
PubMed1979

Clinical correlations of partial deficiency of lipoamide dehydrogenase.

Reduced activities of lipoamide dehydrogenase (LAD) relative to cytochrome oxidase have been found in 12 or 26 patients with inherited ataxias. One of the 12 patients had adult-onset ataxia plus ragged-red muscle fibers. The other 11 had Friedreich syndrome or early-onset variants of this, as did 6 patients with normal enzyme activity. However, the 11 patients with reduced enzyme activity were clinically more homogeneous than the 6 with normal activity.

Dihydrolipoamide Dehydrogenase↗
PubMed1978

Lipoamide dehydrogenase in cultured human skin fibroblasts.

Lipoamide dehydrogenase was identified in cultured skin fibroblasts of normal individuals and patients with Friedreich's ataxia. The optimum conditions for its assay were defined. Data disclosed a normal range of 36--122 mumol/min/mg protein in control fibroblasts and 61--112 mumol/min/mg protein in patients fibroblasts. Numerous precautions should be taken in handling fibroblast cultures for lipoamide dehydrogenase determination.

Buffers↗
PubMed1990

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.

Animals↗
PubMed1989

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.

Amino Acid Oxidoreductases↗
PubMed1983

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties.

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.

Amino Acids↗