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)

A photoaffinity analog of 4-hydroxycoumarin containing an azidobenzyl group at the 3-position and, if desired, carbon-14 or tritium radionuclides has been synthesized and characterized. This compound, 3-(p-azidobenzyl)-4-hydroxycoumarin, serves as an effective competitive inhibitor of the dicoumarol-sensitive NAD(P)H:quinone reductase (EC 1.6.99.2; DT-diaphorase) from rat liver, having an apparent inhibition constant of 6.6 x 10(-8) M, a value comparable to that observed for dicoumarol (1.7 x 10(-9) M), significantly lower than for Warfarin (3.5 x 10(-5) M) and well within the range required of an effective photoaffinity reagent. Irradiation of the reductase with ultraviolet light in the presence of the photoprobe resulted in the covalent labeling of up to 10% of the protein. Greater than 99% of the covalent incorporation is precluded by the addition of 15 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme by this photoaffinity reagent. Further evidence of a high degree of specificity is provided by the isolation and sequence analysis of the peptides covalently modified by this reagent. A single region within the protein was found to be labeled, with threonine 127 and tyrosine 128 being the only amino acid residues that were observed to be modified. These results, for the first time, define a portion of the 4-hydroxycoumarin binding site within a protein that has a well established sensitivity to this type of anticoagulant and, because dicoumarol serves as a competitive inhibitor for pyridine nucleotides in this enzyme, may also define a portion of this unusual pyridine nucleotide binding site. In addition, these results suggest that this reagent may be effective as a highly specific photoaffinity probe in the identification of other proteins that are similarly inhibited by 4-hydroxycoumarin derivatives, such as the microsomal enzymes associated with the vitamin K-dependent carboxylation system.
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PMID:Synthesis of the photoaffinity probe 3-(p-azidobenzyl)-4-hydroxycoumarin and identification of the dicoumarol binding site in rat liver NAD(P)H:quinone reductase (EC 1.6.99.2). 170 34

DT-diaphorase (EC 1.6.99.2), also referred to as NAD(P)H:(quinone-acceptor) oxidoreductase, is involved in the reductive activation process of several cytotoxic antitumor quinones and nitrobenzenes. It has been observed in our and other laboratories that the rat enzyme is significantly more effective in activating these drugs than the human and mouse enzymes. These results indicate that the available cytotoxic drugs are better substrates for the rat enzyme and are not the most ideal prodrugs for activation by DT-diaphorase in human tumors. In this study, using site-directed mutagenesis to replace residues in the rat enzyme with the human sequences and residues in the human enzyme with the rat sequences, we have found that residue 104 (Tyr in the rat enzyme and Gln in the human and mouse enzymes) is an important residue responsible for the catalytic differences between the rat and the human (and mouse) enzymes. With an exchange of a single amino acid, the rat mutant Y104Q behaved like the wild-type human enzyme, and the human mutant Q104Y behaved like the wild-type rat enzyme in their ability to reductively activate the cytotoxic drug CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The study also confirms the conclusion of the x-ray structural analysis of rat enzyme that residue 130 (Thr in the rat enzyme and Ala in the human and mouse enzymes) is positioned near the binding region of the nicotinamide portion of NAD(P)H. This structural information is very important for designing suitable drugs and approaches for human cancer chemotherapy mediated by DT-diaphorase.
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PMID:Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse. 899 9

Na(+)-translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS-PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow-green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. N-Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.
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PMID:Covalently bound flavin in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus. 1083 78

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

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an enzyme that detoxifies quinones and reduces oxidative stress. A cysteine-to-threonine (C --> T) substitution polymorphism at nucleotide 609 of the NQO1 complementary DNA (NQO1 C609T) results in a lowering of NQO1 activity. Individuals homozygous for this mutation have no NQO1 activity, and heterozygotes have low to intermediate activity compared with people with wild type. DNA samples from 493 adult de novo acute leukemia patients and 838 matched controls were genotyped for NQO1 C609T. The majority of cases were diagnosed as acute myeloid leukemia (AML) (n = 420); 67 as acute lymphoblastic leukemia (ALL); and 6 as other forms of acute leukemia. The frequency of cases with low or null NQO1 activity (heterozygote + homozygous mutant) was significantly higher among total acute leukemia case subjects compared with their matched controls (odds ratio [OR] = 1.49; 95% confidence interval [CI], 1.17-1.89). Both ALL (OR = 1.93; 95% CI, 0.96-3.87) and AML case subjects (OR = 1.47; 95% CI, 1.13-1.90) exhibited a higher frequency of low or null NQO1 genotypes than controls. For de novo AML, the most significant effect of low or null NQO1 activity was observed among the 88 cases harboring translocations and inversions (OR = 2.39; 95% CI, 1.34-4.27) and was especially high for those harboring inv(16) (OR = 8.13; 95% CI, 1.43-46.42). These findings were confirmed in a second group of 217 de novo AML cases with known cytogenetics. Thus, inheritance of NQO1 C609T confers an increased risk of de novo acute leukemia in adults, implicating quinones and related compounds that generate oxidative stress in producing acute leukemia.
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PMID:Low NAD(P)H:quinone oxidoreductase 1 activity is associated with increased risk of acute leukemia in adults. 1122 89

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

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

Commonly used antitumor agents, such as DNA topoisomerase I/II poisons, kill cancer cells by creating nonrepairable DNA double-strand breaks (DSBs). To repair DSBs, error-free homologous recombination (HR), and/or error-prone nonhomologous end joining (NHEJ) are activated. These processes involve the phosphatidylinositol 3'-kinase-related kinase family of serine/threonine enzymes: ataxia telangiectasia mutated (ATM), ATM- and Rad3-related for HR, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ. Alterations in these repair processes can cause drug/radiation resistance and increased genomic instability. beta-Lapachone (beta-lap; also known as ARQ 501), currently in phase II clinical trials for the treatment of pancreatic cancer, causes a novel caspase- and p53-independent cell death in cancer cells overexpressing NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 catalyzes a futile oxidoreduction of beta-lap leading to reactive oxygen species generation, DNA breaks, gamma-H2AX foci formation, and hyperactivation of poly(ADP-ribose) polymerase-1, which is required for cell death. Here, we report that beta-lap exposure results in NQO1-dependent activation of the MRE11-Rad50-Nbs-1 complex. In addition, ATM serine 1981, DNA-PKcs threonine 2609, and Chk1 serine 345 phosphorylation were noted; indicative of simultaneous HR and NHEJ activation. However, inhibition of NHEJ, but not HR, by genetic or chemical means potentiated beta-lap lethality. These studies give insight into the mechanism by which beta-lap radiosensitizes cancer cells and suggest that NHEJ is a potent target for enhancing the therapeutic efficacy of beta-lap alone or in combination with other agents in cancer cells that express elevated NQO1 levels.
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PMID:Nonhomologous end joining is essential for cellular resistance to the novel antitumor agent, beta-lapachone. 1763 5

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone pool. It contains one [4Fe-4S] (2+,1+) and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S] (2+,1+) to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S] (+) at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 and -30 mV for wild type to -11 and -19 mV, respectively. The N338A mutation decreased the potentials to -37 and -49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF 1e (-) catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone but not in electron transfer from ETF to ETF-QO. Therefore, the iron-sulfur cluster is the immediate acceptor from ETF.
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PMID:The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein. 1867 1

A wide array of dietary phytochemicals have been reported to induce the expression of enzymes involved in both cellular antioxidant defenses and elimination/inactivation of electrophilic carcinogens. Induction of such cytoprotective enzymes by edible phytochemicals largely accounts for their cancer chemopreventive and chemoprotective activities. Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a crucial role in the coordinated induction of those genes encoding many stress-responsive and cytoptotective enzymes and related proteins. These include NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, glutamate cysteine ligase, glutathione S-transferase, glutathione peroxidase, thioredoxin, etc. In resting cells, Nrf2 is sequestered in the cytoplasm as an inactive complex with the repressor Kelch-like ECH-associated protein 1 (Keap1). The release of Nrf2 from its repressor is most likely to be achieved by alterations in the structure of Keap1. Keap1 contains several reactive cysteine residues that function as sensors of cellular redox changes. Oxidation or covalent modification of some of these critical cysteine thiols would stabilize Nrf2, thereby facilitating nuclear accumulation of Nrf2. After translocation into nucleus, Nrf2 forms a heterodimer with other transcription factors, such as small Maf, which in turn binds to the 5'-upstream CIS-acting regulatory sequence, termed antioxidant response elements (ARE) or electrophile response elements (EpRE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes. Certain dietary chemopreventive agents target Keap1 by oxidizing or chemically modifying one or more of its specific cysteine thiols, thereby stabilizing Nrf2. In addition, phosphorylation of specific serine or threonine residues present in Nrf2 by upstream kinases may also facilitate the nuclear localization of Nrf2. Multiple mechanisms of Nrf2 activation by signals mediated by one or more of the upstream kinases, such as mitogen-activated protein kinases, phosphatidylionositol-3-kinase/Akt, protein kinase C, and casein kinase-2 have recently been proposed. This review highlights the cytoprotective gene expression induced by some representative dietary chemopreventive phytochemicals with the Nrf2-Keap1 system as a prime molecular target.
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PMID:Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. 1893 64


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