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)

Cytosolic NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) accounted for about 68% of the total ubiquinone (UQ) reductase activity in rat liver homogenate [Takahashi, T. et al. (1995) Biochem. J. 309, 883-890]. We investigated the effects of various factors on this enzyme activity in rat liver cytosol with the aim of elucidating its physiological roles. The NADPH-UQ reductase in rat liver cytosol catalyzed the reduction of UQ to UQH2 with concomitant oxidation of equimolar NADPH. The optimal pH was around 7.4, and the optimal temperatures were about 28 degrees C for NADH and about 37 degrees C for NADPH. NADH, deamino NADH, and deamino NADPH were much less active hydrogen donors than NADPH, whereas reduced nicotinamide mononucleotide, ascorbate, erythorbate, reduced glutathione, and cysteine were inactive. As the hydrogen acceptor, UQ-9 had the highest Vmax/Km among the long-chain UQ homologues tested. FAD and FMN stimulated the activity. Anionic detergents, Mg2+ and Sr2+ also enhanced the activity. Rotenone, malonic acid, antimycin A, and KCN, which inhibit mitochondrial and microsomal electron transfer enzymes, superoxide dismutase, and acetylated cytochrome c had no effect on the NADPH-UQ reductase activity. These results indicated that the NADPH-UQ reductase in rat liver cytosol is a flavoprotein that reduces UQ-10 by a two-electron reduction mechanism and is distinguishable from known microsomal and mitochondrial enzymes, as well as DT-diaphorase [EC 1.6.99.2].
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PMID:Characterization of NADPH-dependent ubiquinone reductase activity in rat liver cytosol: effect of various factors on ubiquinone-reducing activity and discrimination from other quinone reductases. 888 15

The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic N-terminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2x[2Fe-2S] and 6x[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.
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PMID:H(+)-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans. Studies on topology and stoichiometry of the peripheral subunits. 1049 27

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD*-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.
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PMID:One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain. 1092 3

The Na(+)-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.
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PMID:FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus. 1116 85

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.
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PMID:Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus. 1124 87

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.
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PMID:Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi. 1140 80

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.
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PMID:Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH. 1152 20

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K(m) 3 microM) and activity with this substrate was inhibited by the presence of dicoumarol (K(i) 1.0 microM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K(m) 40 microM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21.5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K(m) for CB 1954 was lower than that of NTR (617 microM compared to 862 microM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).
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PMID:Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954. 1178 22

The actinomycete Rhodococcus opacus MR11 harbors a bidirectional NAD-reducing [NiFe] hydrogenase (SH). This cytoplasmic enzyme is composed of two heterodimeric modules which catalyze distinct enzymatic activities. The hydrogenase moiety mediates H(2):benzyl viologen oxidoreductase activity and the FMN-containing diaphorase module displays NADH:benzyl viologen oxidoreductase activity. The SH of Rh. opacus resembles [NiFe] hydrogenases present in strains of the proteobacterium Ralstonia eutropha and in species of cyanobacteria. Heterologous expression of active [NiFe] hydrogenases failed in most cases due to protein-assisted maturation processes implicated in the assembly of the NiFe bimetallic site. This study reports on the construction of a recombinant plasmid harboring the four SH subunit genes hoxFUYH and the associated endopeptidase gene hoxW from Rh. opacus under the regime of the SH promoter from R. eutropha H16. The resulting recombinant plasmid restored lithoautotrophic growth in a R. eutropha mutant impaired in H(2)-oxidizing ability. The SH of Rh. opacus was functionally active in R. eutropha and displayed the typical features described for its natural host. It readily dissociated in vitro into two active subforms. Dissociation was accompanied by the loss of the H(2)-dependent NAD-reducing activity, which was partially reconstituted by addition of 5 mM MgSO(4) and 0.5 mM NiCl(2). Activity and stability of the SH from Rh. opacus were enhanced almost three-fold by co-overexpression of the SH-associated metal insertion genes hypA2B2F2 of R. eutropha. Under optimal conditions the heterologously expressed Rh. opacus SH catalyzed NAD-reduction at a specific activity of 1.7 units per mg protein, which is approximately 30% of the yield obtained for the R. eutropha SH. The results indicate that, despite an enormous phylogenetic distance of the two bacterial species, their SH proteins are highly related.
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PMID:Expression of a functional NAD-reducing [NiFe] hydrogenase from the gram-positive Rhodococcus opacus in the gram-negative Ralstonia eutropha. 1180 65

Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is strongly inhibited by a new antibiotic korormicin. Korormicin specifically inhibits the Na(+)-dependent reaction of the NQR complex and acts as a purely non-competitive inhibitor for Q-1 with the inhibitor constant of 82 pM. Korormicin-resistant mutants were isolated from V. alginolyticus and the NQR complex was purified from a mutant KR2. Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin acted as a purely noncompetitive inhibitor to the NQR complex from the mutant KR2, but the inhibitor constant increased to 8 microM, which is 10(5)-fold higher than that of the wild-type NQR complex. The inhibitor constant of HQNO, however, was only slightly affected by the acquisition of korormicin resistance. The spontaneous mutation was caused by a single mutation of G-422 to T-422 in the nucleotide sequence of the nqrB gene, which resulted in the conversion of Gly-140 to Val-140. Thus, Gly-140 seems to play an important role for the binding of korormicin to the NqrB subunit. The fact that korormicin is a purely noncompetitive inhibitor for Q-1 strongly supports the presence of one of Q-1 binding sites in the NqrB subunit, which also has a covalently bound FMN at Thr-235.
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PMID:Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na(+)-translocating NADH-quinone reductase. 1205 67


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