Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis.
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An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential. (+info)
Construction of a homologous selectable marker gene for Lentinula edodes transformation.
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We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 microg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold. (+info)
Effect of the systemic fungicide carboxin on electron transport function in membranes of Micrococcus denitrificans.
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The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) inhibited oxidation of succinate by membranes prepared from Micrococcus denitrificans, the K(i) being 16 muM. Oxycarboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide), F831 (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide), and another succinate oxidase inhibitor, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTB) were less effective inhibitors of succinate oxidation by membranes of M. denitrificans. Oxidation of other substrates (nicotinamide adenine dinucleotide, reduced form, d-lactate, l-lactate, malate, and d,l-alpha-hydroxybutyrate) was inhibited to a lesser degree by carboxin, and formate oxidation was entirely resistant. With all substrates tested, oxycarboxin, the dioxide analogue of carboxin, was less effective than carboxin. Carboxin also inhibited dichlorophenol indophenol (DCIP) reductase activities by these membranes in a manner both qualitatively and quantitatively similar to the inhibition of oxidation of the various substrates. The inhibition of DCIP reductase activities by TTB was qualitatively similar to carboxin, but TTB was a less effective inhibitor with all substrates tested. The inhibition of DCIP reductase by carboxin could be relieved by phenazine methosulfate with all substrates except d-lactate. Only slight inhibition of d-lactate-stimulated uptake of [(14)C]glycine by these membrane vesicles was seen with carboxin. Uptake of [(14)C]glycine could be stimulated to varying degrees with the other substrates tested, but in no case did carboxin cause significant inhibition. Membranes isolated from M. denitrificans are a useful system for investigating the mechanism of inhibition of electron transport function by carboxin, and the use of this system for evaluations of carboxin and its metabolites is suggested. (+info)
3-nitropropionic acid is a suicide inhibitor of mitochondrial respiration that, upon oxidation by complex II, forms a covalent adduct with a catalytic base arginine in the active site of the enzyme.
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We report three new structures of mitochondrial respiratory Complex II (succinate ubiquinone oxidoreductase, E.C. 1.3.5.1) at up to 2.1 A resolution, with various inhibitors. The structures define the conformation of the bound inhibitors and suggest the residues involved in substrate binding and catalysis at the dicarboxylate site. In particular they support the role of Arg(297) as a general base catalyst accepting a proton in the dehydrogenation of succinate. The dicarboxylate ligand in oxaloacetate-containing crystals appears to be the same as that reported for Shewanella flavocytochrome c treated with fumarate. The plant and fungal toxin 3-nitropropionic acid, an irreversible inactivator of succinate dehydrogenase, forms a covalent adduct with the side chain of Arg(297). The modification eliminates a trypsin cleavage site in the flavoprotein, and tandem mass spectroscopic analysis of the new fragment shows the mass of Arg(297) to be increased by 83 Da and to have the potential of losing 44 Da, consistent with decarboxylation, during fragmentation. (+info)
Oxathiin carboxanilide, a potent inhibitor of human immunodeficiency virus reproduction.
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Oxathiin carboxanilide (OC), NSC 615985, a compound originally synthesized as a potential fungicide, was demonstrated to be highly active in preventing human immunodeficiency virus (HIV)-induced cell killing and in inhibiting HIV reproduction. Virus-infected CD4+ lymphocytes were completely protected by 0.5 microM OC, whereas no toxicity was observed at concentrations below 50 microM OC. Production of infectious virus, viral p24 antigen, and virion reverse transcriptase were reduced by OC at concentrations that prevented viral cell killing. A variety of CD4+ T-cell lines were protected by OC from HIV cytopathicity, and OC inhibited two distinct strains of HIV-1. However, HIV-2 infections were unaffected by OC. OC had no direct effect on virions of HIV or on the enzymatic activities of HIV reverse transcriptase or HIV protease. Time-limited treatments of cells with OC before, during, or after exposure of cells to virus failed to protect cells from the eventual cytopathic effects of HIV, and OC failed to inhibit the production of virus from cells in which infection was established or from chronically infected cells. We conclude that the highly active OC has a reversible effect on some early stage of HIV-1 reproduction and cytopathicity. Pilot in vivo experiments showed that circulating concentrations of OC exceeding 1 microM could be achieved and sustained in hamsters for at least a week with no remarkable toxicological sequelae. OC represents a new class of anti-HIV agents that are promising candidates for drug development. (+info)
Structure of Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-binding site.
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Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides.
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Five carboxin-resistant mutants from Aspergillus oryzae were characterized by the sensitivities of their mycelial growth and succinate dehydrogenase (SDH) activity to carboxin and three related fungicides. Despite a significant resistance to carboxin, exhibited by all the mutants, their patterns of sensitivity to the other fungicides was distinct. This provides clues to the molecular interaction between SDH and these fungicides. (+info)
Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple.
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