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 carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
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PMID:Probing the role of the carboxyl-terminal region of ferredoxin-NADP+ reductase by site-directed mutagenesis and deletion analysis. 836 77

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.
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PMID:Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction. 841 79

To investigate the functional role of the cysteine residues present in the spinach ferredoxin-NADP+ oxidoreductase, we individually replaced each of the five cysteine residues with serine using site-directed mutagenesis. All of the mutant reductases were correctly assembled in Escherichia coli except for the C42S mutant protein. C114S and C137S mutant enzymes apparently showed structural and kinetic properties very similar to those of the wild-type reductase. However, C272S and C132S mutations yielded enzymes with a decreased catalytic activity in the ferredoxin-dependent reaction (14 and 31% of the wild type, respectively). Whereas the C132S was fully competent in the diaphorase reaction, the C272S mutant flavoprotein showed a 35-fold reduction in catalytic efficiency with respect to the wild-type enzyme (0.4 versus 14.28 microM-1 s-1) due to a substantial decrease of kcat. NADP+ binding by the C272S mutant enzyme was apparently quantitatively the same (Kd = 37 microM) but qualitatively different, as shown by the differential spectrum. Stopped-flow experiments showed that the enzyme-FAD reduction rate was considerably decreased in the C272S mutant reductase, along with a much lower yield of the charge-transfer transient species. It is inferred from these data that the charge transfer (FAD-NADPH) between the reductase and NADPH is required for hydride transfer from the pyridine nucleotide to flavin to occur with a rate compatible with catalysis.
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PMID:The role of cysteine residues of spinach ferredoxin-NADP+ reductase As assessed by site-directed mutagenesis. 851 83

Using non-denaturing gel electrophoresis and staining with nitro-blue tetrazolium, we reveal the presence of two NAD(P)H oxidoreductase activity bands within thylakoids membranes of Solanum tuberosum L. Second dimension SDS-PAGE and Western analysis show that one of the activity bands contains several polypeptides, two of them being recognized by antibodies directed against peptides corresponding to conserved domains of chloroplastic genes products NDH B and NDH J (at 32 and 18 kDa, respectively). Both activity bands also contain a polypeptide (around 36 kDa) recognized by an antibody directed against ferredoxin-NADP(+)-reductase (FNR). We conclude from these results that both chloroplastic ndh B and ndh J gene products are components of a thylakoid NAD(P)H dehydrogenase complex. The association with FNR is suggested to allow the complex to use NADPH instead of NADH as a preferential substrate.
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PMID:Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase complex. 855 17

Neurons exhibiting reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity (NADPHd) were quantified at 500 microns rostrocaudal intervals in spinal trigeminal nucleus (Vsp) of adenalectomized (ADX), ADX + corticosterone, and sham-ADX rats 6-12 days after surgery. NADPHd neurons were found predominantly in Vsp subnucleus caudalis (Vc) and in dorsomedial subnucleus oralis. ADX significantly increased the number of NADPHd neurons in superficial laminae of Vc, an effect reversed by chronic corticosterone replacement. ADX effects on NADPHd in superficial laminae of Vc but not in deep laminae of Vc or in the periobex region of Vsp paralleled previously observed sites of ADX enhancement of noxious stimulus-induced Fos-like immunoreactivity. The results indicate that chronic changes in adrenal steroid status regulate NADPHd, a mechanism that may both derive from changes in nitric oxide synthase expression and influence the processing of nociceptive information by central trigeminal neurons.
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PMID:Adrenalectomy increases reduced nicotinamide adenine dinucleotide phosphate-diaphorase activity in the rat spinal trigeminal subnucleus caudalis. 870 97

MX100 is an Escherichia coli K12 genotoxicity tester strain, especially developed for mechanistic studies of chemical mutagens and carcinogens. For the study of the role of specific enzymes in the bioactivation and bioinactivation of carcinogens, it is necessary to characterize MX100 as far as its metabolic bio(in)activation capacities are concerned. In this study such a characterization is performed in two types of cell-free lysates, one derived from stationary phase cells, grown in rich medium (SR-lysates) and one from exponentially growing cells (log phase), cultured in minimal medium (LM-lysates). Six Phase I enzyme activities of aromatic NADPH hydroxylase, NADH hydroxylase, flavin-containing monooxygenase (FMO), nitroreductase, DT-diaphorase and NADPH ferredoxin:oxidoreductase were determined. Activities of six Phase II enzymes glutathione S-transferases (GSTs), N-aryl acetyltransferase (NAT), arylamine sulphotransferase, UDP-glucuronyltransferase and epoxide hydratase and of the Phase III enzyme cysteine conjugate beta-lyase were subsequently assessed. In addition, five antioxidant enzymes: superoxide dismutase (SOD), catalase, glutathione (GSH)-reductase, GSH-peroxidase and alkyl hydroperoxide reductase; as well as concentrations of glutathione (GSH) and its disulphide (GSSG), were measured. The activity parameters of all enzymes were compared with those obtained in similar lysates of the Salmonella strain TA100 and in rat liver preparations. The results indicate that MX100 as well as TA100 contain relatively low oxidative but high reductase Phase I activities. Both strains demonstrated low activities for the Phase II conjugation enzymes except for GSTs. In MX100, relatively high activities were detected for all antioxidative enzymes, activities which were lower in TA100. Significant differences in activities were observed between the SR-lysates derived from stationary phase/rich medium and LM-lysates from log phase/minimal medium cells for nitroreductase, GST, SOD, catalase, NADPH ferredoxin:oxidoreductase as well as in GSH content. In general, we described for the first time a metabolic characterization of the E.coli tester strain MX100 and the Salmonella typhimurium strain TA100 and discussed the results in terms of its significance for carcinogen bioactivation and bioinactivation capacities.
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PMID:Characterization of enzyme activities and cofactors involved in bioactivation and bioinactivation of chemical carcinogens in the tester strains Escherichia coli K12 MX100 and Salmonella typhimurium LT2 TA100. 923 69

We have synthesized nitroaromatic derivatives of triterpenoid betulin (lup-20(29)-ene-3 beta, 28-diol), betulin-(28)-5'-(aziridin-1-yl)-2',4'-dinitrobenzoate and betulin-(28)-5'-nitro-2'-furoate. These compounds were reduced in single-electron way by ferredoxin: NADP+ reductase and flavocytochrome b2 at rates comparable with their simple structure analogs. Besides, these compounds were substrates for DT-diaphorase. Their toxicity to bovine leukemia virus-transformed lamb fibroblast culture was partly prevented by antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, indicating an involvement of oxidative stress in their cytotoxicity.
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PMID:Nitroaromatic betulin derivatives as redox cycling agents. 923 38

We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.
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PMID:Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity. 934 69

Ferredoxin and ferredoxin-NADP+ reductase are the two last partners of the photosynthetic electron-transfer chain, responsible for the final reduction of NADP+ to NADPH. Herein, we report the engineering and characterization of a novel protein molecule in which the electron-carrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The gene was obtained by joining the cDNAs encoding the respective proteins and subsequently by deleting the intervening sequence between them by site-directed mutagenesis. No extra amino acid residues were introduced between the C-terminus of ferredoxin I and the N-terminus of the flavoenzyme. The chimera was purified to homogeneity and characterized. The M(r) of the chimera apoprotein was 45,800 as determined by mass spectrometry, in agreement with the expected value of 45,846. Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprotein. The chimera was found active as a diaphorase and, more interestingly, highly efficient as a cytochrome c reductase, without need for free ferredoxin addition in the assay medium. Several lines of evidence indicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological complex. Thus, the fusion protein could be a useful tool for studying the mechanism of protein-protein recognition and electron transfer in the ferredoxin-ferredoxin-NADP+ reductase system.
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PMID:A three-domain iron-sulfur flavoprotein obtained through gene fusion of ferredoxin and ferredoxin-NADP+ reductase from spinach leaves. 939 97

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.
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PMID:Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis. 951 8


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