A novel aspect of the inhibition by arsenicals of binding-protein-dependent galactose transport in gram-negative bacteria.

The inhibitory effects of arsenate and arsenite on binding-protein-dependent transport systems are reconsidered. It is shown that arsenate inhibits binding-protein-dependent galactose transport in proteoliposomes energized either by dihydrolipoamide and NAD+ or by a membrane potential (under conditions where ATP metabolism is not implicated); this result is in contradiction with the current interpretation of arsenate inhibition of binding-protein-dependent transport systems (which is based on ATP depletion) and can be explained by reference to the recently discovered ATP inhibition of the binding-protein-dependent galactose transport. In whole cells, the greater inhibition by arsenate of lipoamide-dependent transport than of protonmotive-force-dependent transport may be explained by a modification by arsenate of the pools of several compounds metabolized by 2-oxo-acid dehydrogenases (which have been implicated in binding-protein-dependent transport). The inhibition of binding-protein-dependent galactose transport by arsenite is probably linked to the inhibition by arsenite of the galactose-stimulated lipoamide dehydrogenase activity implicated in this transport and is reminiscent of the known arsenite inhibition of lipoamide dehydrogenases.     

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.     

Remodulation of central carbon metabolic pathway in response to arsenite exposure in Rhodococcus sp. strain NAU‐1

Arsenite‐tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU‐1 (aerobic, Gram‐positive, non‐motile, coccobacilli), was hyper‐tolerant to arsenite (As^III, 23 mM) and arsenate (As^V, 180 mM). 16S rRNA gene of NAU‐1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU‐1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S‐transferase) increased in dose‐dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in As^III concentration. Metabolic studies revealed that Rhodococcus NAU‐1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose‐6‐phosphate dehydrogenase, pyruvate dehydrogenase and α‐ketoglutarate dehydrogenase enzymes respectively, in the presence of As^III. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU‐1 in the presence of As^III.     

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.     

Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings

Plants reconfigure their metabolic network under stress conditions. Changes of mitochondrial metabolism such as tricarboxylic acid (TCA) cycle and amino acid metabolism are reported in Arabidopsis roots but the exact molecular basis underlying this remains unknown. We here hypothesise the reassembly of enzyme protein complexes to be a molecular mechanism for metabolic regulation and tried in the present study to find out mitochondrial protein complexes which change their composition under oxidative stress by the combinatorial approach of proteomics and metabolomics. Arabidopsis seedlings were treated with menadione to induce oxidative stress. The inhibition of several TCA cycle enzymes and the oxidised NADPH pool indicated the onset of oxidative stress. In blue native/SDS-PAGE analysis of mitochondrial protein complexes the intensities of 18 spots increased and those of 13 spots decreased in menadione treated samples suggesting these proteins associate with, or dissociate from, protein complexes. Some spots were identified as metabolic enzymes related to central carbon metabolism such as malic enzyme, glyceraldehyde-3-phosphate dehydrogenase, monodehydroascorbate reductase and alanine aminotransferase. The change in spot intensity was not directly correlated to the total enzyme activity and mRNA level of the corresponding enzyme but closely related to the metabolite profile, suggesting the metabolism is regulated under oxidative stress at a higher level than translation. These results are somewhat preliminary but suggest the regulation of the TCA cycle, glycolysis, ascorbate and amino acid metabolism by reassembly of plant enzyme complexes.     

Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

The genome of the yeast Saccharomyces cerevisiae encodes a canonical lipoamide dehydrogenase (Lpd1p) as part of the pyruvate dehydrogenase complex and a highly similar protein termed Irc15p (increased recombination centers 15). In contrast to Lpd1p, Irc15p lacks a pair of redox active cysteine residues required for the reduction of lipoamide and thus it is very unlikely that Irc15p performs a similar dithiol‐disulfide exchange reaction as reported for lipoamide dehydrogenases. We expressed IRC15 in Escherichia coli and purified the produced protein to conduct a detailed biochemical characterization. Here, we show that Irc15p is a dimeric protein with one FAD per protomer. Photoreduction of the protein generates the fully reduced hydroquinone without the occurrence of a flavin semiquinone radical. Similarly, reduction with NADH or NADPH yields the flavin hydroquinone without the occurrence of intermediates as observed for lipoamide dehydrogenase. The redox potential of Irc15p was ?313 ± 1 mV and is thus similar to lipoamide dehydrogenase. Reduced Irc15p is oxidized by several artificial electron acceptors such as potassium ferricyanide, 2,6‐dichlorophenol‐indophenol, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide, and menadione. However, disulfides such as cystine, glutathione, and lipoamide were unable to react with reduced Irc15p. Limited proteolysis and SAXS‐measurements revealed that the NADH‐dependent formation of hydrogen peroxide caused a substantial structural change in the dimeric protein. Therefore, we hypothesize that Irc15p undergoes a conformational change in the presence of elevated levels of hydrogen peroxide, which is a putative biomarker of oxidative stress. This conformational change may in turn modulate the interaction of Irc15p with other key players involved in regulating microtubule dynamics.     

Identification of mutants in phosphorus metabolism

Phosphorus availability is often limiting for plant growth. However, little is known of the pathways and mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed a screen based on the induction of secreted root acid phosphatase activity by low‐P stress to identify mutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detected visually in the roots of A. thaliana seedlings grown in vitro on low‐P medium, using the chromogenic substrate, 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (BCIP). In low‐P stress conditions the roots of wild‐type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the coloured product. Potential mutants were identified as having white, or pale blue, roots under these conditions. Out of approximately 79 000 T‐DNA mutagenised seedlings screened, two mutants with reduced acid phosphatase staining were further characterised. Both exhibited reduced growth and differences in their P contents when compared to wild‐type A. thaliana. The mutant with the most severe phenotype, pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within the leaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants (phol and pho2) and from an acid phosphatase deficient mutant (pupl) of A. thaliana. This suggested that the screening method was robust and might lead to the identification of further mutants with the potential for increasing our understanding of P nutrition.     

Leishmania infantum: provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)₂] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)₂ can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)₂ is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.     

Characterization of dihydrolipoamide dehydrogenase from the mitochondria of Helicoverpa armigera,a pest resistant to insecticides

Dihydrolipoamide dehydrogenase (DHLDH) was isolated from the mitochondria of Helicoverpa armigera, a destructive pest which has developed resistance to commonly used insecticides. The flavoenzyme was purified 17.98‐fold to homogeneity with an overall yield of 10.53% by employing ammonium sulfate precipitation, hydroxylapatite chromatography and CM‐Sephadex chromatography. The purified enzyme exhibited the specific activity of 18.7 U/mg and was characterized as a dimer with a subunit mass of 66 kDa. The enzyme showed specificity for nicotinamide adenine dinucleotide – hydrogen (NADH) and lipoamide, as substrates, with Michaelis‐Menten constants (K_m) of 0.083 mmol/L and 0.4 mmol/L, respectively. The reduction reaction of lipoamide by the enzyme could be explained by ping‐pong mechanism. The spectra of DHLDH showed the maximum absorbance at 420 nm, 455 nm and 475 nm. The enzyme activity was strongly inhibited by mercurial and arsenical compounds. The N‐terminal sequence of Ha‐DHLDH showed homology with those of mammalian and arthropod DHLDH. Since H. armigera has developed high levels of resistance to commonly used insecticides, biochemical properties of the metabolic enzymes such as DHLDH, could be helpful to develop insecticidal molecules for the control of H. armigera, with a different mode of action.     

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.     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.     

A lipoamide dehydrogenase from Neisseria meningitidis has a lipoyl domain

A protein of molecular weight of 64 kDa (p64k) found in the outer membrane of Neisseria meningitidis shows a high degree of homology with both the lipoyl domain of the acetyltransferase and the entire sequence of the lipoamide dehydrogenase, the E2 and E3 components of the dehydrogenase multienzyme complexes, respectively. The alignment of the p64k with lipoyl domains and lipoamide dehydrogenases from different species is presented. The possible implications of this protein in binding protein-dependent transport are discussed. This is the first lipoamide dehydrogenase reported to have a lipoyl domain. © 1995 Wiley-Liss, Inc.     

Effects of Denervation and Simple Disuse on Rates of Oxidation and on Activities of Four Mitochondrial Enzymes in Type I Muscle

To differentiate the effect of muscle contractile activity from that of motor nerve on oxidative processes in type I muscle, oxidative processes were studied in muscle after immobilization and after denervation. The two processes led to similar atrophy of muscle weight and of the mean diameter of muscle fibers. Disuse of soleus muscle (type I) did not affect rates of oxidation of 14C-labeled substrates although these were reduced by disuse of the vastus lateralis (type II). Disuse of the soleus did not affect activities of several mitochondrial enzymes assayed by histochemical or biochemical methods. However, denervation of the soleus did lead to a fall in metabolic rates and enzyme activities. The activity of 3-hydroxybutyrate dehydrogenase fell more than did the activities of succinic dehydrogenase, lipoamide dehydrogenase, or cytochrome-c oxidase in both homogenates and in mitochondrial fractions. These results suggest nerve may regulate mitochondrial enzymes in type I muscle. The mechanism appears to be different from that which regulates oxidative processes in type II muscle.     

pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.     

A novel pathway of arsenate detoxification

Microorganisms have evolved various mechanisms to detoxify arsenic, an ubiquitous environmental toxin. Known mechanisms include arsenite efflux, arsenate reduction followed by arsenite efflux and arsenite methylation. In this issue, Chen et al. describe a novel mechanism for arsenate detoxification via synergistic interaction of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and a major facilitator superfamily protein (ArsJ). They propose that GAPDH catalyzes the formation of 1‐arseno‐3‐phosphoglycerate, which is then extruded out of the cell by ArsJ. The significance of this pathway and questions for further research are discussed.     

Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis

Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T‐DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co‐expressed with a number of genes encoding mitochondrial proteins, including SDH1‐1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin‐adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein‐bound flavin‐adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro‐respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Development of a downstream process for the isolation of Staphylococcus aureus arsenate reductase overproduced in Escherichia coli

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.     

Broad connections in the Arabidopsis seed metabolic network revealed by metabolite profiling of an amino acid catabolism mutant

An Arabidopsis thaliana mutant was identified as having increases in 12 of 20 free proteogenic amino acids in seeds. Because these metabolites are produced from multiple, seemingly unrelated biosynthetic networks, it was not possible to use a candidate gene approach to discover the enzyme defect responsible for this complex syndrome. Complementary metabolite profiling analyses revealed increased seed homomethionine and isovaleroyloxypropyl‐glucosinolate, along with reduced 3‐benzoyloxypropyl‐glucosinolate. These data led to the discovery of impaired branched chain amino acid catabolic enzyme isovaleryl‐CoA dehydrogenase (encoded by gene At3g45300 or atIVD) as the cause of this metabolic syndrome. These results indicate that catabolism plays an important role in regulating levels of branched chain amino acids in seeds. The diverse set of metabolites affected in the ivd1 mutants suggests the existence of a more complex network regulating seed amino acid accumulation than previously observed. This combined targeted and non‐targeted metabolite profiling approach is broadly applicable to the characterization of metabolic mutants, human disease studies and crop germplasm.