Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hydrogenosomes are double-membraned ATP-producing and hydrogen-producing organelles of diverse anaerobic eukaryotes. In some versions of endosymbiotic theory they are suggested to be homologues of mitochondria, but alternative views suggest they arose from an anaerobic bacterium that was distinct from the mitochondrial endosymbiont. Here we show that the 51-kDa and 24-kDa subunits of the NADH dehydrogenase module in complex I, the first step in the mitochondrial respiratory chain, are active in hydrogenosomes of Trichomonas vaginalis. Like mitochondrial NADH dehydrogenase, the purified Trichomonas enzyme can reduce a variety of electron carriers including ubiquinone, but unlike the mitochondrial enzyme it can also reduce ferredoxin, the electron carrier used for hydrogen production. The presence of NADH dehydrogenase solves the long-standing conundrum of how hydrogenosomes regenerate NAD+ after malate oxidation. Phylogenetic analyses show that the Trichomonas 51-kDa homologue shares common ancestry with the mitochondrial enzyme. Recruitment of complex I subunits into a H2-producing pathway provides evidence that mitochondria and hydrogenosomes are aerobic and anaerobic homologues of the same endosymbiotically derived organelle.
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PMID:Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I. 1574 82

BphA3 from Pseudomonas sp. KKS102 is a Rieske-type [2Fe-2S] ferredoxin that transfers electrons from an NADH-dependent oxidoreductase, BphA4, to a biphenyl dioxygenase complex. A high-level expression and purification system for the recombinant BphA3 in Escherichia coli was constructed. Two histidine ligands of the Rieske-type cluster in BphA3, were each replaced with serine, cysteine, asparagine and tyrosine. The single mutants, in which either His44 or His65 was replaced with a cysteine residue (CH and HC mutants respectively), and the double mutant, in which both histidine residues were replaced with cysteine residue (CC mutant), accumulated to high levels in the E. coli cells, while the other single mutants did not. The purified WT (wild-type) protein showed characteristic near-UV and visible absorption and CD spectra of Rieske-type clusters. The X-ray absorption spectra were suggestive of the existence of [2Fe-2S] clusters, with one histidine and three cysteine ligands in the CH and HC mutants, and an [2Fe-2S] cluster with four cysteine ligands in the CC mutant. The BphA4-dependent cytochrome c reductase activities of the mutants were less than 0.3% of that of the WT protein. The redox potential of the WT protein determined by cyclic voltammetry was -180+/-5 mV compared with the standard hydrogen electrode, and that of the CH mutant was approx. 175 mV lower. The changes in the near-UV and visible absorption spectra of the mutants showed that the reduced iron-sulphur clusters in the mutants were unstable. His44 and His65 in BphA3 can be replaced with cysteine residues, but are required for the stabilization of the reduced form of the cluster.
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PMID:Tolerance of the Rieske-type [2Fe-2S] cluster in recombinant ferredoxin BphA3 from Pseudomonas sp. KKS102 to histidine ligand mutations. 1573 56

Mitochondrial NADH dehydrogenase (complex I) of plants includes quite a number of plant-specific subunits, some of which exhibit sequence similarity to bacterial gamma-carbonic anhydrases. A homozygous Arabidopsis knockout mutant carrying a T-DNA insertion in a gene encoding one of these subunits (At1g47260) was generated to investigate its physiological role. Isolation of mitochondria and separation of mitochondrial protein complexes by Blue-native polyacrylamide gel electrophoresis or sucrose gradient ultracentrifugation revealed drastically reduced complex I levels. Furthermore, the mitochondrial I + III2 supercomplex was very much reduced in mutant plants. Remaining complex I had normal molecular mass, suggesting substitution of the At1g47260 protein by one or several of the structurally related subunits of this respiratory protein complex. Immune-blotting experiments using polyclonal antibodies directed against the At1g47260 protein indicated its presence within complex I, the I + III2 supercomplex and smaller protein complexes, which possibly represent subcomplexes of complex I. Changes within the mitochondrial proteome of mutant cells were systematically monitored by fluorescence difference gel electrophoresis using 2D Blue-native/SDS and 2D isoelectric focussing/SDS polyacrylamide gel electrophoresis. Complex I subunits are largely absent within the mitochondrial proteome. Further mitochondrial proteins are reduced in mutant plants, like mitochondrial ferredoxin, others are increased, like formate dehydrogenase. Development of mutant plants was normal under standard growth conditions. However, a suspension cell culture generated from mutant plants exhibited clearly reduced growth rates and respiration. In summary, At1g47260 is important for complex I assembly in plant mitochondria and respiration. A role of At1g47260 in mitochondrial one-carbon metabolism is supported by micro-array analyses.
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PMID:Disruption of a nuclear gene encoding a mitochondrial gamma carbonic anhydrase reduces complex I and supercomplex I + III2 levels and alters mitochondrial physiology in Arabidopsis. 1593 78

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O(2) uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H(2) in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H(2) production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H(2) production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.
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PMID:Inhibitor studies on non-photochemical plastoquinone reduction and H(2) photoproduction in Chlamydomonas reinhardtii. 1595 Sep 24

Metronidazole and related 5-nitroimidazoles are the only available drugs in the treatment of human urogenital trichomoniasis caused by the protozoan parasite Trichomonas vaginalis. The drugs are activated to cytotoxic anion radicals by their reduction within the hydrogenosomes. It has been established that electrons required for metronidazole activation are released from pyruvate by the activity of pyruvate:ferredoxin oxidoreductase and transferred to the drug by a low-redox-potential carrier, ferredoxin. Here we describe a novel pathway involved in the drug activation within the hydrogenosome. The source of electrons is malate, another major hydrogenosomal substrate, which is oxidatively decarboxylated to pyruvate and CO2 by NAD-dependent malic enzyme. The electrons released during this reaction are transferred from NADH to ferredoxin by NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I, which uses ferredoxin as electron acceptor. Trichomonads acquire high-level metronidazole resistance only after both pyruvate- and malate-dependent pathways of metronidazole activation are eliminated from the hydrogenosomes.
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PMID:Alternative pathway of metronidazole activation in Trichomonas vaginalis hydrogenosomes. 1630 69

The present study investigated the hypothalamic gene expressions regulated by glucocorticoids (GC), key hormones in energy homeostasis. Using the serial analysis of gene expression (SAGE) method, we studied the effects of adrenalectomy (ADX) and GC on the transcriptomes of mouse hypothalamus. Approximately 180,000 SAGE tags, which correspond to 50,000 tag species, were isolated from each group of intact or adrenalectomized mice as well as 1, 3, and 24 h after GC injection. ADX upregulated diazepam binding inhibitor gene expression while downregulating vomeronasal 1 receptor D4, genes involved in mitochondrial phosphorylation (cytochrome-c oxidase 1 and NADH dehydrogenase 3), 3beta-hydroxysteroid dehydrogenase-1, and prostaglandin D2 synthase. GC increased the gene expression levels of dehydrogenase/reductase member 3, prostaglandin D2 synthase, solute carrier family 4 member 4, and five cytoskeletal proteins including myosin light chain phosphorylatable fast and troponin C2 fast. On the other hand, GC reduced the mRNA levels of calmodulin 1 and expressed sequence tag similar to calmodulin 2, ATP synthase F0 subunit 6, and solute carrier family 4 member 3. Moreover, 7 uncharacterized and 43 novel transcripts were modulated by ADX and GC. The present study has identified genes that may regulate hypothalamic systems governing energy balance in response to ADX and GC.
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PMID:Regulation of hypothalamic gene expression by glucocorticoid: implications for energy homeostasis. 1636 73

A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region to contain a tetraCys motif and treatment with biarsenical fluorescein. Fluorescence anisotropy was used to study the protein-protein interactions among various combinations of the four components of the complex. Binding interactions were detected between T4moD and the hydroxylase component T4moH [K(D) value of 83 nM for interaction with the alphabetagamma protomer] and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC (K(D) value of 78 nM). No binding interactions were detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF was able to disrupt binding between T4moC and T4moD. The detected binding interactions suggest an intermediary electron transfer complex between T4moC and T4moD that excludes T4moF. The results indicate that specialization of effector protein function may include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.
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PMID:Component interactions and implications for complex formation in the multicomponent toluene 4-monooxygenase. 1663 29

Chemical modification of spinach chloroplasts by phenylglyoxal and dansyl chloride resulted in inhibition of NADP photoreduction. The rate of inactivation was higher with both reagents when modification was carried out in the light with methylviologen or phenazine methosulfate present. Uncouplers prevent the effect of light. Electron transport from water to methylviologen was not affected by the modifiers.The presence of 10 millimolar NADP completely protected the membrane-bound reductase against inactivation by phenylglyoxal. With lower concentrations, protection was higher in the light than in the dark. The apparent dissociation constants of the enzyme-substrate complex for NADP were 0.9 and 0.1 millimolar for the dark and light inactivation, respectively. Inactivation of NADP photoreduction by dansyl chloride was completely prevented by ferredoxin, but only partially by nucleotides.The diaphorase activity was inhibited in chloroplasts modified by phenylglyoxal, but not when modified by dansyl chloride.The results suggest that energizing thylakoid membranes by light induces a conformational change in membrane-bound ferredoxin-NADP reductase, and that the reductase is an allotopic enzyme.
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PMID:Effect of Light on Chemical Modification of Chloroplast Ferredoxin-NADP Reductase. 1666 Dec 21

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.Young lambsquarters plants were treated with atrazine by spraying the leaves at a rate which was lethal for susceptible plants after 5 or 6 days, but had little effect on the resistant biotype. Nitrite did not accumulate in either biotype, but remained present at the level of about 0.1 microgram nitrite N per gram fresh weight. The nitrate content of susceptible-type leaves did increase to two or three times the initial level, during the first four days after spraying. Usually the only visible effect on the plants during this time was a decreased growth rate. Twenty-four hours after spraying the following activities had fallen to 25% or less of the activities of solvent-sprayed control plants: in vivo nitrite reductase, in vivo nitrate reductase, in vitro NADH-nitrate reductase, in vitro reduced flavin mononucleotidenitrate reductase, and in vitro NADH-diaphorase. In these atrazine-treated plants, in vitro nitrite reductase activity with reducing potential supplied by methylviologen was not affected, nor were any of the above activities in leaves of atrazine-treated resistant plants. The abrupt fall in nitrate reductase represents an effect of atrazine not directly related to inhibition of photosynthesis.
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PMID:Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity. 1666 20

The binding of ferredoxin-NADP reductase to spinach chloroplast membranes was studied by washing the membranes with different media. Release of the enzyme from the thylakoids was greater in 0.75 millimolar EDTA but was not complete inasmuch as 20% the activity remained membrane-bound after three washes.A Scatchard plot of binding experiments suggests the presence of one type of binding site and a stoichiometry of 3 to 4 nanomoles of reductase per micromole of chlorophyll was calculated. Rebinding has a nonspecific requirement for cations. Their effectiveness increased with their valency. Rebinding of purified enzyme to depleted membranes resulted in a stimulation of its diaphorase activity.It is suggested that binding of ferredoxin-NADP reductase to thylakoid membranes is dependent upon neutralization of negative charges.
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PMID:Interaction of Ferredoxin-NADP Oxidoreductase with the Thylakoid Membrane. 1666 60


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