Superoxide anion production by lipoamide dehydrogenase redox-cycling: effect of enzyme modifiers.

Redox-cycling of porcine heart lipoamide dehydrogenase in the presence of NADH and oxygen produced O2-. (NADH-oxidase activity) as demonstrated by (a) reduction of cytochrome c; (b) reduction of the Fe(III)-ADP complex; (c) lucigenin luminescence and (d) the inhibitory effect of superoxide dismutase. NAD+ and p-chloromercuribenzoate inhibited O2-. generation whereas arsenite enhanced it. Comparison of heart and yeast enzyme preparations revealed a close correlation between lipoamide reductase and NADH-oxidase activities. It is concluded that O2-. production is a molecular property of lipoamide dehydrogenase.     

Multifunctionality of lipoamide dehydrogenase: activities of chemically trapped monomeric and dimeric enzymes

Lipoamide dehydrogenase from pig heart exists in monomer-dimer equilibrium. The effect of the state of subunit aggregation on the multifunctionality of lipoamide dehydrogenase was investigated by the use of chemically trapped monomeric and dimeric enzymes. Reductive carboxymethylation with 2-mercaptoethanol-iodoacetate yields the stable monomeric enzyme which has been isolated for structural and kinetic studies. The chemically induced monomerization is accompanied by conformational changes resulting in an increased mobility of flavin-adenine dinucleotide. The chemically trapped monomer shows an enhanced diaphorase activity, a reduced electron transferase activity, and a complete loss in dehydrogenase as well as transhydrogenase activities. The enhanced diaphorase activity is associated with increased catalytic efficiencies and the reversal of an inhibitory NADH effect at high concentrations. Treatment of lipoamide dehydrogenase with dimethyl suberimidate gives amidinated samples containing crosslinked dimer. The crosslinked enzyme exhibits a higher dehydrogenase catalytic efficiency than the noncrosslinked enzyme with different kinetic mechanisms without significantly affecting the kinetic parameters of diaphorase reaction. Although the dimeric structure is intimately associated with the dehydrogenase activity, it does not preclude the diaphorase activity. An altered flavin-adenine dinucleotide environment accompanying monomerization is likely responsible for the enhanced diaphorase activity.     

Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.     

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.     

Homotropic regulation of dihydrolipoyl dehydrogenase from bovine kidney

Dihydrolipoyl dehydrogenase from bovine kidney catalyzes NAD-linked redox reaction of lipoamide. Hates of the catalyzed reaction were studied in both directions. Saturation curves for NAD and lipoamide are nonhyperbolic, suggesting homotropic cooperative interactions of these substrates with the enzyme. The cooperative effect was analyzed by Hill plots according to the diagnostic procedure of Levitzki and Koshland. Dihydrolipoyl dehydrogenase is subject to homotropic regulation in which NAD acts as a negative cooperative effector, whereas lipoamide acts as a positive cooperative effector. At high concentrations, dihydrolipoamide normalizes the saturation curve of NAD, while NADH tends to enhance the cooperative interaction of lipoamide with the enzyme.     

Role of Lipoamide Dehydrogenase and Metallothionein on 1-Methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced Neurotoxicity

In the present study, we investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on lipoamide dehydrogenase activity and metallothionein content. Lipoamide dehydrogenase is a flavoprotein enzyme, which reduces lipoamide and low molecular weight thiols. This enzyme has also been involved in the conversion of ubiquinone (coenzyme Q-10, oxidized form) to ubiquinol (reduced form). Lipoamide dehydrogenase activity was measured spectrophotometrically following its incubation with different doses of MPTP, MPP⁺, and divalent metals. MPTP at higher concentrations inhibited the lipoamide dehydrogenase activity, whereas it’s potent toxic metabolite 1-methyl-4-phenylpyridinium (MPP⁺) had a similar effect at lower concentration. Calcium and copper did not affect the enzyme activity at any of the doses tested, whereas, zinc dose dependently enhanced the lipoamide dehydrogenase activity. Additionally, levels of metallothionein in the mouse nigrostriatal system were measured by cadmium affinity method following administration of MPTP. Metallothionein content was significantly reduced in the substantia nigra (SN), and not in the nucleus caudatus putamen (NCP) following a single administration of MPTP (30 mg/kg, i.p.). Our results suggests that both lipoamide dehydrogenase activity and metallothionein levels may be critical for dopaminergic neuronal survival in Parkinson’s disease and provides further insights into the neurotoxic mechanisms involved in MPTP-induced neurotoxicity.     

Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. Evidence for a single lipoamide dehydrogenase in plant mitochondria.

In order to purify the lipoamide dehydrogenase associated with the glycine decarboxylase complex of pea leaf mitochondria, the activity of free lipoamide dehydrogenase has been separated from those of the pyruvate and 2-oxoglutarate dehydrogenase complexes under conditions in which the glycine decarboxylase dissociates into its component subunits. This free lipoamide dehydrogenase which is normally associated with the glycine decarboxylase complex has been further purified and the N-terminal amino acid sequence determined. Positive cDNA clones isolated from both a pea leaf and embryo lambda gt11 expression library using an antibody raised against the purified lipoamide dehydrogenase proved to be the product of a single gene. The amino acid sequence deduced from the open reading frame included a sequence matching that determined directly from the N terminus of the mature protein. The deduced amino acid sequence shows good homology to the sequence of lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex from Escherichia coli, yeast, and humans. The corresponding mRNA is strongly light-induced both in etiolated pea seedlings and in the leaves of mature plants following a period of darkness. The evidence suggests that the mitochondrial enzyme complexes: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine decarboxylase all use the same lipoamide dehydrogenase subunit.     

Turnover of pegeon breast muscle pyruvate dehydrogenase complex.

The pigeon breast muscle pyruvate dehydrogenase complex was resolved into three component enzymes: lipoate acetyltransferase, pyruvate dehydrogenase, and lipoamide dehydrogenase. The antibodies against each component enzyme were prepared. All of the antibodies against component enzymes precipitated the pyruvate dehydrogenase complex. The enzyme complex was recovered as the immunoprecipitate from the extract of breast muscle of a pigeon that had received a single injection of L-[4,5-3H]leucine. The immunoprecipitate was separated into each component enzyme by SDS-polyacrylamide gel electrophoresis. The relative isotopic leucine incorporations per mg of protein into each component enzyme 4 h after the injection were 1.0 : 0.9 : 1.4 : 2.7 for lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase, respectively. The half-lives of lipoate acetyltransferase, alpha- and beta-subunit of pyruvate dehydrogenase, and lipoamide dehydrogenase were 7.7, 2.5, 2.6, and 1.8 days, respectively. These results indicate that the component enzymes of the pyruvate dehydrogenase complex were synthesized and degraded at different rates.     

Interaction of selenite and tellurite with thiol-dependent redox enzymes: Kinetics and mitochondrial implications

The interactions of selenite and tellurite with cytosolic and mitochondrial thioredoxin reductases (TrxR1 and TrxR2) and glutathione reductases (GR) from yeast and mammalian sources were explored. Both TrxR1 and TrxR2 act as selenite and tellurite reductases. Kinetic treatment shows that selenite has a greater affinity than tellurite with both TrxR1 and TrxR2. Considering both k_cat and K_m, selenite shows a better catalytic efficiency than tellurite with TrxR1, whereas with TrxR2, the catalytic efficiency is similar for both chalcogens. Tellurite is a good substrate for GR, whereas selenite is almost completely ineffective. Selenite or tellurite determine a large mitochondrial permeability transition associated with thiol group oxidation. However, with increasing concentrations of both chalcogens, only about 25% of total thiols are oxidized. In isolated mitochondria, selenite or tellurite per se does not stimulate H₂O₂ production, which, however, is increased by the presence of auranofin. They also determine a large oxidation of mitochondrial pyridine nucleotides. In ovarian cancer cells both chalcogens decrease the mitochondrial membrane potential. These results indicate that selenite and tellurite, interacting with the thiol-dependent enzymes, alter the balance connecting pyridine nucleotides and thiol redox state, consequently leading to mitochondrial and cellular alterations essentially referable to a disulfide stress.     

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

In vivo 31P nuclear magnetic resonance investigation of tellurite toxicity in Escherichia coli

Here we compare the physiological state of Escherichia coli exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed Escherichia coli HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants tehAB. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by 31P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by 31P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant tehAB, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.     

Radial diffusion assay of lipoamide dehydrogenase and its use to assess riboflavin deficiency.

A new, simple, and quantitative method was developed to determine lipoamide dehydrogenase (E,C, 1.6,4.3, NADH:lipoamide oxidoreductase). The principle of this technique is to allow the enzyme or tissue extracts to diffuse in an agarose gel containing lipoate. The enzyme, after 24 hr of diffusion and 2 hr of reaction with NADH, can be determined by the size of a dark or fluorescence-quenching zone in the gel when illuminated with uv light. The diameter of the quenching zone which indicates the enzymatic oxidation of NADH is linearly proportional to the logarithm of enzyme concentration. Since lipoamide dehydrogenase is a FAD-enzyme, the activity was decreased in liver homogenates of riboflavin-deficient chicks, as measured by the new method. This demonstrated the potential importance of this new technique in nutritional and clinical applications.     

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.     

Immunochemical comparison of lipoamide dehydrogenases from various sources and reactivity of various lipoamide dehydrogenases with rat heart pyruvate dehydrogenase-subcomplex

Lipoamide dehydrogenases from various sources were purified and their immunochemical properties were compared. Antibody against rat lipoamide dehydrogenase reacted with rat, human, pig, pigeon and frog enzymes, but not with enzymes from E. coli, yeast and Ascaris. Anti-Ascaris enzyme and anti-E. coli enzyme antibodies reacted with Ascaris and E. coli enzymes, respectively. The pyruvate dehydrogenase subcomplex, which consists of pyruvate dehydrogenase and lipoate acetyltransferase, was prepared by releasing the lipoamide dehydrogenase from rat heart pyruvate dehydrogenase complex by anti-lipoamide dehydrogenase antibody. Lipoamide dehydrogenases from various sources were added to rat pyruvate dehydrogenase subcomplex and the complex overall activity was measured. Each lipoamide dehydrogenase effectively recovered the overall activity of rat pyruvate dehydrogenase subcomplex to 80% of the original activity.     

Glutathione is a target in tellurite toxicity and is protected by tellurite resistance determinants in Escherichia coli

Tellurite (TeO3(2-)) is highly toxic to most microorganisms. The mechanisms of toxicity or resistance are poorly understood. It has been shown that tellurite rapidly depletes the reduced thiol content within wild-type Escherichia coli. We have shown that the presence of plasmid-borne tellurite-resistance determinants protects against general thiol oxidation by tellurite. In the present study we observe that the tellurite-dependent depletion of cellular thiols in mutants of the glutathione and thioredoxin thiol:redox system was less than in wild-type cells. To identify the type of low-molecular-weight thiol compounds affected by tellurite exposure, the thiol-containing molecules were analyzed by reverse phase HPLC as their monobromobimane derivatives. Results indicated that reduced glutathione is a major initial target of tellurite reactivity within the cell. Other thiol species are also targeted by tellurite, including reduced coenzyme A. The presence of the tellurite resistance determinants kilA and ter protect against the loss of reduced glutathione by as much as 60% over a 2 h exposure. This protection of glutathione oxidation is likely key to the resistance mechanism of these determinants. Additionally, the thiol oxidation response curves were compared between selenite and tellurite. The loss of thiol compounds within the cell recovered from selenite but not to tellurite.     

Lipoamide dehydrogenase from Azotobacter vinelandii. The role of the C-terminus in catalysis and dimer stabilization.

The 10 C-terminal residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C-terminus folds back towards the active site and is involved in interactions with the other subunit. The function of the C-terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme delta 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme delta 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16-mutated enzymes is comparable to enzyme delta 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. vinelandii enzyme. In the delta 9 and delta 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C-terminus containing residues 450-470.