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:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (DeltaHf(ArNO(2)(-*)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP(+) reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in DeltaHf(ArNO(2)(-*)). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their DeltaHf(ArNO(2)(-*)). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.
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PMID:Quantitative structure-activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity. 1151 95

Ferredoxin and ferredoxin-NADP+ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.
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PMID:Ferredoxin and ferredoxin-NADP reductase from photosynthetic and nonphotosynthetic tissues of tomato. 1153 2

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.
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PMID:Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. 1207 65

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(alpha2betagamma)4; molecular mass 600 +/- 50 kDa] composed of a propionyl-CoA dehydrogenase (alpha2, 2 x 40 kDa) and an electron-transferring flavoprotein (ETF; beta, 38 kDa; gamma, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per alpha2betagamma subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 +/- 1 microm; kcat = 4.5 s-1 at 100 microm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 microm; kcat = 29 s-1 at 300 microm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 microm; kcat = 2.0 s-1) or butyryl-CoA (Km = 100 microm; kcat = 3.5 s-1) as electron donor and 200 microm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the beta and gamma subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.
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PMID:Acryloyl-CoA reductase from Clostridium propionicum. An enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein. 1260 23

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.
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PMID:The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase. 1457 60

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.
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PMID:Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity. 1511 Nov 26

The cytochrome b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2-4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH poly-peptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)-1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300-350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the "lipid augmentation" technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 A.
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PMID:Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis. 1518 70

From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM. Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.
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PMID:Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC. 1525 6

With an aim to understand the toxicity mechanisms of the explosive 4,6-dinitro- benzofuroxan (DNBF), we studied its single-electron reduction by NADPH:cytochrome P450 reductase and ferredoxin:NADP(+) reductase, and two- electron reduction by DT-diaphorase and Enterobacter cloacae nitroreductase. The enzymatic reactivities of DNBF and another explosive 2,4,6-trinitrotoluene (TNT) were similar, except for the much lower reactivity of DNBF towards nitroreductase. DNBF was less cytotoxic in FLK cells than TNT. However, their action shared the same mechanisms, oxidative stress and activation by DT-diaphorase. The lower cytotoxicity of DNBF may be explained by the negative electrostatic charge of its adduct with water which may impede cellular membrane penetration, and by the formation of its less reactive adducts with intracellular reduced glutathione.
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PMID:Enzymatic redox reactions of the explosive 4,6-dinitrobenzofuroxan (DNBF): implications for its toxic action. 1562 81

Dissipation mechanisms of excess photon energy under water stress were studied in ndhB-inactivated tobacco (Nicotiana tabacum cv. Xanthi) mutants, which are impaired in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI. Relative leaf water content and net CO(2) assimilation decreased to 30% and almost zero, respectively, after 11-day water stress in the mutant and wild type plants. Similar reductions in PSII activity (by ca. 75%), and increases in malondialdehyde (by ca. 45%), an indicator of lipid peroxidation, were observed in both the plant groups when subjected to water stress. The stressed mutant and wild type plants showed similar P700 redox kinetics, but only the stressed mutant demonstrated an enhanced operation of the antimycin A-sensitive, ferredoxin-dependent cyclic electron flow around PSI, as indicated by a transient increase in chlorophyll fluorescence after turning off of actinic light. Further, the stressed mutant showed higher oxidation of alpha-tocopherol to alpha-tocopherol quinone, as compared with that in the stressed wild type. Thus, a deficiency in NAD(P)H dehydrogenase-dependent cyclic electron flow around PSI does not lead to oxidative damage because the mutant compensates for this deficiency by activating alternative dissipating routes of excess photon energy, such as up-regulation of ferredoxin-dependent cyclic electron flow around PSI and increased accumulation of alpha-tocopherol quinone.
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PMID:Enhanced ferredoxin-dependent cyclic electron flow around photosystem I and alpha-tocopherol quinone accumulation in water-stressed ndhB-inactivated tobacco mutants. 1591 57


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