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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene encoding the streptococcal flavoprotein NADH oxidase (NOXase), which catalyzes the four-electron reduction of O2-->2H2O, has been cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The deduced NOXase protein sequence corresponds to a molecular mass of 48.9 kDa and contains three previously sequenced cysteinyl peptides obtained with the purified enzyme. In Escherichia coli, the expressed nox gene produced a catalytically active product, which retained its immunoreactivity to affinity-purified NOXase antisera. Alignment of the NOXase protein sequence with that of streptococcal NADH peroxidase (NPXase) revealed that the proteins are 44% identical. Among the most highly conserved segments is a sequence containing Cys42; this residue is known to exist as a stabilized cysteine-sulfenic acid (Cys-SOH) in NPXase and serves as the non-flavin redox center. In addition, three previously identified NPXase segments, known to be involved in FAD and NAD(P)-binding in other pyridine nucleotide-linked flavoprotein oxidoreductases, are strongly conserved in NOXase. Overall, the extensive homology observed between NOXase and NPXase suggests that the monomer chain fold of the oxidase closely resembles that of the peroxidase. Both sequences share limited but significant homology to those of glutathione reductase and other members of the flavoprotein disulfide reductase family. These and other considerations suggest that these two unusual streptococcal flavoproteins constitute a distinct class of FAD-dependent oxidoreductases, the flavoprotein peroxide reductases, easily contrasted with enzymes such as glutathione reductase and thioredoxin reductase.
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PMID:Molecular cloning and analysis of the gene encoding the NADH oxidase from Streptococcus faecalis 10C1. Comparison with NADH peroxidase and the flavoprotein disulfide reductases. 140 82

Thioredoxin is a small oxidation-reduction (redox) mediator protein. Its reduction by NADPH is catalyzed by the flavoenzyme thioredoxin reductase. Site-directed mutagenesis has provided forms of the reductase in which Cys135 and Cys138 have each been changed to a serine residue (Prongay, A. J., Engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). Cys135 and Cys138 form the redox-active disulfide in the oxidized enzyme. The redox properties of the two altered forms of Escherichia coli thioredoxin reductase have been determined from pH 6.0 to 9.0. Photoreduction of TRR(Ser135,Cys138) produces the blue, neutral semiquinone species, which disproportionates (Kf = 0.73) to an apparent maximum of 29% of the total enzyme as the semiquinone. In contrast, the semiquinone formed on TRR(Cys135,Ser138) during a photoreductive titration does not disproportionate and 70% of the enzyme is stabilized as the semiquinione. Reductive titrations have demonstrated that 1 mol of sodium dithionite (2 electrons)/mol of FAD is required to fully reduce TRR(Ser135,Cys138) whereas 2 mol of dithionite/mol of FAD are required to fully reduce TRR(Cys135,Ser138). The oxidation-reduction midpoint potentials for the 1-electron and 2-electron reductions of TRR(Ser135,Cys138) have been determined by NADH/NAD+ titrations in the presence of a mediator, benzyl viologen. The midpoint potential for the 2-electron reduction of TRR(Ser135,Cys138) is -280 mV, at pH 7.0 and 20 degrees C. Thus, the redox potential is similar to that of the FAD/FADH2 couple in the dithiol form of wild type enzyme, -270 mV (corrected to 20 degrees C) (O'Donnell, M. E., and Williams, C. H., Jr. (1983) J. Biol. Chem. 258, 13795-13805). The delta Em/delta pH is -57.1 mV, which corresponds to a proton stoichiometry of 2 H+/2 e-.A maximum of 19% of the enzyme forms a stable semiquinone species during the titration, and the potentials for the oxidized enzyme/semiquinone couple, E2, and the semiquinone/reduced enzyme couple, E1, are -306 and -256 mV, respectively, at pH 7.0 and 20 degrees C. These studies provide evidence that the residue at position 138 exerts a greater effect on the FAD than does the residue at position 135.
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PMID:Oxidation-reduction properties of Escherichia coli thioredoxin reductase altered at each active site cysteine residue. 146 18

Quinoids undergo metabolism by a number of flavoenzymes. Reactive species formed during the metabolism of some quinoids might be anticipated to inhibit flavoenzyme activity. Several quinoids have been tested for their ability to inhibit rat liver thioredoxin reductase (TR). The antitumor quinones diaziquone and doxorubicin, and the quinoneimine 2,6-dichloroindophenol, were found to be inhibitors of the reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) by TR. The inhibition was most marked after incubation of the quinoid with NADPH and the enzyme for 60 min before adding DTNB, with Ki values of 0.5 microM for diaziquone, 0.5 microM for doxorubicin, and 0.07 microM for 2,6-dichloroindophenol. The three quinoids all produced a time-dependent and first order loss of TR activity. There was formation of electron spin resonance-detectable semiquinoid free radicals upon incubation of diaziquone, doxorubicin and 2,6-dichloroindophenol with TR and NADPH under anaerobic conditions. Oxygen radicals formed by redox cycling of the quinoids did not make a major contribution to the inhibition of TR by the quinoids, as shown by the absence of significant reversal of the inhibition by anaerobic incubation conditions and the lack of effect of the oxygen radical scavengers dimethyl sulfoxide, superoxide dismutase and catalase. It was not possible to demonstrate NADPH-dependent covalent binding of radiolabeled diaziquone or doxorubicin to the TR apoprotein. It is possible that the quinoids bind noncovalently to the enzyme apoprotein, or bind to the FAD prosthetic group. The results of the study suggest that some antitumor quinoids are mechanism-based inhibitors of TR showing metabolism- and time-dependent irreversible inhibition of enzyme activity.
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PMID:Mechanism-based inhibition of thioredoxin reductase by antitumor quinoid compounds. 156 82

DNA fragments encoding streptococcal NADH peroxidase (NPXase) have been amplified, cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The NPXase gene (npr) comprises 1341 base-pairs and is preceded by a typical ribosome binding site. Upstream from the structural gene, putative -10 and -35 promoter regions have been identified, as has a possible factor-independent terminator that occurs in 3'-flanking sequences. The deduced relative molecular mass (Mr = 49,551), amino acid composition and isoelectric point of NPXase are in good agreement with previous values obtained with the purified enzyme. In addition, three sequenced peptides totaling approximately 20% of the protein were located in the npr gene product. From the sequencing data the deduced NPXase sequence shares low but significant homology with the flavoprotein disulfide reductase class of enzymes ranging from 21% for glutathione reductase (GRase) to 28% for thioredoxin reductase. Alignment of NPXase to Escherichia coli GRase allowed the identification of three previously reported fingerprints for the FAD, NADP+ and central domains of GRase, in the peroxidase sequence. In addition, Cys42 of NPXase, which is present as an unusual stabilized cysteine-sulfenic acid in the oxidized enzyme, aligns favorably with the charge-transfer cysteine in E. coli GRase, and both residues closely follow FAD-binding folds found near their respective amino termini. Such sequence characteristics can also be seen in mercuric reductase, lipoamide dehydrogenase and trypanothione reductase, suggesting that all these enzymes may have originally diverged from a common ancestor. Sequences that are on average 50% identical with three previously reported peptides of the related streptococcal NADH oxidase were also identified in the NPXase primary structure, suggesting a strong similarity between these flavoenzymes. Using the E. coli phage T7 expression system the npr gene has now been overexpressed in an E. coli genetic background. The resultant overexpressing clone produced a recombinant NPXase that was catalytically active and immunoreactive to NPXase antisera.
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PMID:Cloning, sequence and overexpression of NADH peroxidase from Streptococcus faecalis 10C1. Structural relationship with the flavoprotein disulfide reductases. 171 12

The DNA sequence of the Salmonella typhimurium ahp locus was determined. The locus was found to contain two genes that encode the two proteins (C22 and F52a) that comprise the S. typhimurium alkyl hydroperoxide reductase activity. The predicted sequence of the F52a protein component of the alkyl hydroperoxide reductase was found to be highly homologous to the Escherichia coli thioredoxin reductase protein (34% identity with many conservative substitutions). The homology was found to be particularly striking in the region containing the redox-active cysteines of the thioredoxin reductase molecule, and among the identities were the redox-active cysteines themselves. Aside from the strong similarity to thioredoxin reductase, overall homology between the F52a protein and other flavoprotein disulfide oxidoreductases such as glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase was found to be rather limited, and the conserved active site segment common to the three proteins was not observed within the F52a protein. However, three short segments that have been implicated in FAD and NAD binding were found to be conserved between the F52a protein and the other disulfide reductases. These results suggest that the alkyl hydroperoxide reductase is the second known member of a class of disulfide oxidoreductases which was represented previously by thioredoxin reductase alone; they also allow the putative assignment of several functional domains.
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PMID:Alkyl hydroperoxide reductase from Salmonella typhimurium. Sequence and homology to thioredoxin reductase and other flavoprotein disulfide oxidoreductases. 219 51

The flavoenzyme thioredoxin reductase from Escherichia coli contains an oxidation-reduction active disulfide made up of Cys135 and Cys138. Mutations changing each Cys residue to a Ser residue have been effected (Prongay, A. J., engelke, D. R., and Williams, C. H., Jr. (1989) J. Biol. Chem. 264, 2656-2664). The FAD prosthetic group of each altered thioredoxin reductase has been replaced with 1-deaza-FAD (a flavin analog with carbon substituted for nitrogen at position 1), 4-thio-FAD (a flavin analog with sulfur substituted for oxygen at position 4), and 6-thiocyanato-FAD. 1-Deaza-FAD-TRR(Cys135,Ser138) has absorbance and fluorescence spectral properties similar to the oxidized form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD. The absorbance spectrum of 1-deaza-FAD-TRR(Ser135,Cys138) is similar to the spectrum of the two-electron reduced form of wild type apothioredoxin reductase reconstituted with 1-deaza-FAD, indicating that it is a mixture of two species (O'Donnell, M. E., and Williams, C. H., Jr. (1984) J. Biol. Chem. 259, 2243-2251). The spectrum of one of these species of 1-deaza-FAD-TRR(Ser135,Cys138) resembles the spectrum of oxidized 1-deaza-FAD bound to wild type apothioredoxin reductase. The other species has an absorbance spectrum with a single peak at 400 nm (epsilon 400 = 11,100 M-1 cm-1) and resembles the spectrum of a thiolate adduct at the C4a position of the 1-deaza-FAD. The equilibrium between these species is pH-dependent, with a maximum of 50% C4a-adduct formation at low pH, and is linked to pK alpha values at 8.2 and 9.3. The absorbance spectrum of 4-thio-FAD-TRR(Cys135,Ser138) resembles the spectrum of the unbound 4-thio-FAD, whereas 4-thio-FAD-TRR(Ser135,Cys138) has a spectrum indicative of a mixture of 4-thio-FAD and FAD, suggesting a reaction between the 4-position of the flavin and Cys138. The binding of 6-thiocyanato-FAD to the apoprotein of the mutated enzymes showed no evidence for a reaction between the thiols and the group at the 6-position of the flavin.
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PMID:Evidence for direct interaction between cysteine 138 and the flavin in thioredoxin reductase. A study using flavin analogs. 222 55

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5'-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine X HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 +/- 0.6 microM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity.
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PMID:Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y. 243 Aug 4

Thioredoxin reductase (TRR), a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, undergoes two sequential thiol-disulfide interchange reactions with thioredoxin during catalysis. In order to assess the catalytic role of each nascent thiol of the active site disulfide of thioredoxin reductase, the 2 cysteines (Cys-136 and Cys-139) forming this disulfide have been individually changed to serines by site-directed mutageneses of the cloned trxB gene of Escherichia coli. Spectral analyses of TRR(Ser-136,Cys-139) as a function of pH and ionic strength have revealed two pKa values associated with the epsilon 456, one of which increases from 7.0 to 8.3 as the ionic strength is increased, and a second at 4.4 which is seen only at high ionic strength. epsilon 458 of wild type TRR(Cys-136,Cys-139) and epsilon 453 of TRR(Cys-136,Ser-139) are pH-independent. A charge transfer complex (epsilon 530 = 1300 M-1 cm-1), unique to TRR(Ser-136,Cys-139), has been observed under conditions of high ammonium cation concentration (apparent Kd = 54 microM) at pH 7.6. These results suggest the assignment of Cys-139 as the FAD-interacting thiol in the reduction of thioredoxin by NADPH via thioredoxin reductase. If, as with other members of this enzyme family, the two distinct catalytic functions are each carried out by a different nascent thiol, then Cys-136 would perform the initial thiol-disulfide interchange with thioredoxin. Steady state kinetic analyses of the proteins have revealed turnover numbers of 10 and 50% of the value of the wild type enzyme for TRR(Ser-136,Cys-139) and TRR(Cys-136,Ser-139), respectively, and no changes in the apparent Km values of TR(S2) or NADPH. The finding of activity in the mutants indicates that the remaining thiol can carry out interchange with the disulfide of thioredoxin, and the resulting mixed disulfide can be reduced by NADPH via the flavin.
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PMID:Characterization of two active site mutations of thioredoxin reductase from Escherichia coli. 264 68

The DNA sequence of the Escherichia coli gene encoding thioredoxin reductase has been determined. The predicted protein sequence agrees with an earlier determination of the 17 amino-terminal amino acids and with a fragment of the protein containing the redox-active half-cystines. Similarity between E. coli thioredoxin reductase and other flavoprotein disulfide oxidoreductases is quite limited, but three short segments, two of which are probably involved in FAD and NADPH binding, are highly conserved between thioredoxin reductase, glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase.
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PMID:Sequence of thioredoxin reductase from Escherichia coli. Relationship to other flavoprotein disulfide oxidoreductases. 328 28

Azelaic acid has been shown to inhibit thioredoxin reductase (TR) at the surface of guinea pig and human skin, on cultures of human keratinocytes, melanocytes, melanoma cells, murine melanoma cells (Cloudman S91), and on purified enzymes from Escherichia coli, rat liver, and human melanoma. Human melanoma cells are more resistant to inhibition by azelaic acid than murine melanoma or human melanocytes. Kinetic studies with pure TRs indicate that azelaic acid is a reversible competitive inhibitor. Fluorescence spectroscopy has been used to show that azelaic acid does not interfere with electron transfer from NADPH to FAD on TR. However, azelaic acid does inhibit electron transfer from the dithiolate active site of this enzyme. Inhibition by azelaic acid is pH-dependent, requiring the dissociation of both carboxylate groups, and also the dissociation of the active site dithiol groups. Binding studies with [14C]azelaic acid at different pHs, indicate that inhibition is first due to the formation of a thioester on the active thiolate groups followed by transacylation of a basic amino acid residue in the active site. A comparative study of TR inhibition by C6, C9, C10 and C12 saturated dicarboxylic acids was also determined on guinea pig skin in vivo. These homologous dicarboxylic acids gave greater inhibition with increasing size (i.e. mol wt.).
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PMID:Azelaic acid as a competitive inhibitor of thioredoxin reductase in human melanoma cells. 365 30


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