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)

Mercuric ion reductase (the merA gene product) is a unique member of the class of FAD and redox-active disulfide-containing oxidoreductases by virtue of its ability to reduce Hg(II) to Hg(0) as the last step in bacterial detoxification of mercurials. In addition to the active site redox-active disulfide, formed between Cys135 and Cys140 in Tn501 MerA, the protein products of the three merA gene sequences published to date have two additional conserved pairs of cysteines, one near the N-terminus (Cys10Cys13 in Tn501 MerA) and another near the C-terminus (Cys558Cys559 in Tn501 MerA). Neither of these pairs is found in other members of this enzyme family. To assess the possible roles of these peripheral cysteines in the Hg(II) detoxification pathway, we have constructed and characterized one single mutant, Cys10Ala13, and two double mutants, Ala10Ala13 and Ala558Ala559. The N-terminal mutants are fully functional in vivo as determined by HgCl2 resistance studies, showing the N-terminal cysteine pair to be dispensable. In contrast, the Ala558Ala559 mutant is defective for HgCl2 resistance in vivo and Hg(SR)2 reduction in vitro, thereby implicating Cys558 and/or Cys559 in Hg(II) reduction by the wild-type enzyme. Other activities, such as NADPH/thio-NADP+ transhydrogenation, NADPH oxidation, and DTNB reduction, are unimpaired in this mutant.
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PMID:Mutagenesis of the N- and C-terminal cysteine pairs of Tn501 mercuric ion reductase: consequences for bacterial detoxification of mercurials. 254 Aug 17

Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.
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PMID:Characterization of fumarate reductase from baker's yeast: essential sulfhydryl group for binding of FAD. 389 9

Effectors and products of enzymatic diiodotyrosine (DIT) deiodination by a cytosolic fraction of pig liver hab been investigated. 13% of the degraded 131I-DIT was found as monoiodotyrosine by thin layer chromatography. The main quantity of the deiodinated DIT was found on the start point of the chromatogram bound to enzyme protein. Tyrosine as a reaction product of enzymatic deiodination of [14C]-IT could not be identified exactly. The liver cytosolic deiodinase is activated by pyruvate; the extent of activation depends on th pyruvate concentration. Diiodohydroxyphenylpyruvate as a product of transamination and theoretically possible intermediate product could be excluded. NADPH 2 and sodium dithionite activated the deiodinase to 1/3, sodium dithionite together with FAD to 1/2 the amount of which was determined for the action of pyruvate. The enzymatic activity in the presence of pyruvate and NADPH2, respectively NADPH2/FAD is identical with the sum of the single activities. The effect of dithionite and sulfite on deiodinase activity depends on the concentration: low effector concentrations increase, while high concentrations decrease the enzyme activity. The liver plasma deiodinase was inactivate quantitatively by reaction with 10(-4) M PCMB; by reaction with 10(-4) M DTNB or NEM the inactivation was 40% only. The inactivation of deiodinase by PCMB was quantitative reversible by cysteine, while inactivation by DTNB was reversible by cysteine to maximal 70% only. Differences between cytosolic and microsomal deiodinases are discussed also in regard to the mechanism of DIT-deiodination by a liver cytosolic fraction with direct participation of SH-groups.
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PMID:[Effectors and products of enzymatic diiodotyrosine deiodination by a plasmatic fraction from pig liver]. 730 42

CDP-6-deoxy-delta 3,4-glucoseen reductase (E3), which catalyzes the reduction of the C-3 deoxygenation step during the formation of CDP-ascarylose, a 3,6-dideoxyhexose found in the lipopolysaccharide of Yersinia pseudotuberculosis, has been expressed at high level in Escherichia coli (670 times over the wild-type strain). This flavoenzyme, which also contains one plant ferredoxin type [2Fe-2S] cluster, was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide. In both cases the inactivation followed a pseudo first order kinetics. The second order rate constant for the reaction of DTNB with E3 was 0.25 mM-1 min-1 at 20 degrees C, pH 8.0. Detailed characterization of the inactivated enzyme showed that neither the flavin nor the [2Fe-2S] cluster was altered during inactivation. Since this inactivation was reversible by treating the inactivated enzyme with 1 mM D,L-dithiothreitol (DTT), it was concluded that only cysteine residues were modified during inactivation. Analysis of the inactivation using the method developed by Tsou revealed that two cysteines react with DTNB at similar rates and modification of either one is enough to impair E3's activity. Tryptic digestion of E3 labeled with N-ethyl[2,3-14C]maleimide, followed by fractionation of the digest by high performance liquid chromatography, gave two labeled peptides, both of which were separately isolated as a pair of interconvertible diastereoisomers. Sequence analysis of these labeled peptides allowed the identification of Cys-75 and Cys-296 as the reactive cysteine residues. Interestingly, the C75S and C296S mutant proteins exhibit identical physical and comparable catalytic properties as the wild-type enzyme. Since Cys-296 is a conserved residue in the NAD(P) binding domain of enzymes belonging to the same class, this residue may be involved in stabilizing the charge-transfer complex between E3 and NADH, thus facilitating hydride transfer from the nicotinamide nucleotide to flavin. A chemically modified Cys-75 which is immediately adjacent to the [2Fe-2S] center in E3 may prevent the proper juxtaposition of the redox centers and thus impede electron transfer leading to enzyme inactivation. These results may be useful for placing constraints on the peptide folding comprising the active site of E3 for electron transfer between NADH, FAD, and the [2Fe-2S] center.
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PMID:Mechanistic studies on CDP-6-deoxy-delta 3,4-glucoseen reductase: the role of cysteine residues in catalysis as probed by chemical modification and site-directed mutagenesis. 770 27

A flavoprotein from Amphibacillus xylanus catalyzes the reduction of oxygen to hydrogen peroxide. Each polypeptide chain in the tetrameric enzyme contains 5 cysteine residues. The complete reduction of enzyme by dithionite requires 6 electrons. Such behavior indicates the presence of redox centers in addition to the FAD, and these could be disulfides. In order to assess the catalytic role of disulfide in the enzyme, 2 of the cysteines (Cys-337 and Cys-340), which show a high degree of homology with alkyl hydroperoxide reductase F52a protein and thioredoxin reductase, have been changed to serines by site-directed mutagenesis of the cloned flavoprotein gene (individually and in a double mutant). Titration of the three mutant enzymes, lacking Cys-337, Cys-340, or both cysteines, requires only 2 electron eq to reach the reduced flavin state. These results indicate the absence of a redox-active disulfide and demonstrate the involvement of Cys-337 and Cys-340 in the redox-active disulfide. The catalytic activity of the three enzymes was examined by steady-state analysis. The Km for NADH and oxygen and the kcat value of these mutant enzymes were essentially the same as those of wild type. The NADH oxidase activities were also accelerated markedly in the presence of free FAD, which is the case for wild-type enzyme. The NADH:5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) oxidoreductase activities of all mutant enzymes were less than 3% of the activity of wild-type enzyme. The weak DTNB reductase activities in the mutant enzymes lacking Cys-337 or Cys-340 may occur through direct reduction of the mixed disulfide Cys-337-thiol or Cys-340-thiol and nitrothiobenzoate by FADH2. However, the weak DTNB reductase activity in the mutant enzyme lacking both cysteines indicates that FADH2 can reduce either DTNB or another disulfide directly, albeit inefficiently. These results suggest intramolecular dithiol-disulfide interchange reactions in the flavoprotein.
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PMID:Role of cysteine 337 and cysteine 340 in flavoprotein that functions as NADH oxidase from Amphibacillus xylanus studied by site-directed mutagenesis. 772 98

Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated. Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45%. In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein. Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E. coli cells. It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture). The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group. Recombinant plant type NTR is as effective as E. coli NTR in the DTNB (5,5'-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different. These results are discussed in relation to the primary structures of NADPH thioredoxin reductases.
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PMID:Arabidopsis thaliana NAPHP thioredoxin reductase. cDNA characterization and expression of the recombinant protein in Escherichia coli. 830

The flavoenzyme thioredoxin reductase (TR) and its natural substrate thioredoxin comprise a redox system generally found in all organisms. In order to better understand the biochemistry of this redox system, TR was purified (> 4000-fold) from human placenta as a dimer of 60-kDa subunits. The molecular size of native TR was determined to be 160 kDa by gel filtration chromatography whereas migration on a sucrose gradient gave a molecular mass of 130 kDa. The pI of TR was determined to be 4.85. The temperature optima for DTNB and insulin reduction by TR were 52 and 40 degrees C, respectively. Preincubation of TR at 60 degrees C for up to 1 h showed no decrease in the enzymatic rates when assayed at 28 degrees C, while temperatures above 65 degrees C resulted in an irreversible loss of activity. Circular dichroism (CD) spectra of TR indicated that the secondary structural changes at 60 degrees C were only partly reversible at 28 degrees C. CD studies showed the flavoenzyme had a TM of 63 degrees C and above 45 degrees C began to exhibit changes in the secondary structure. Equilibrium denaturation of TR by temperature and guanidine hydrochloride suggested that FAD was not displaced during inactivation of TR and that the tertiary structure was primarily disrupted prior to denaturation of the secondary structure. The results of this study show that purified human TR is a relatively thermostable flavoenzyme whose tightly bound FAD group is not displaced by elevated temperatures up to 60 degrees C or by relatively low concentrations of guanidine hydrochloride.
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PMID:Purification of human thioredoxin reductase: properties and characterization by absorption and circular dichroism spectroscopy. 834 16

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI-1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 microM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent Km values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 microM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.
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PMID:A new selenoprotein from human lung adenocarcinoma cells: purification, properties, and thioredoxin reductase activity. 857 4

We describe the purification and characterisation of a thioredoxin reductase-like disulphide reductase from the ancient protozoan parasite, Giardia duodenalis. This dimeric flavoprotein contains 1 mol FAD per subunit and had an apparent subunit molecular mass of 35 kDa. The purified enzyme catalysed the NADPH-dependent (Km = 8 microM) reduction of 5,5'-dithio-bis(2-nitrobenzoic acid) to thionitrobenzoate and was unable to utilise NADH as an electron donor. The sulphydryl-active compounds, N-ethylmaleimide, sodium arsenite and Zn2+ ions, strongly inhibited the enzyme suggesting that a thiol component forms part of the active site. Purified enzyme was able to utilise a variety of substrates, including cystine and oxidised glutathione, which suggests that it is a broad-range disulphide reductase, probably accounting for the majority of thiol cycling activity in this organism. While the G. duodenalis enzyme does not require an intermediate electron transport protein, analogous to thioredoxin, for activity, we have identified a candidate carrier protein which enhances DTNB turnover six fold, therefore implying that Giardia contains a thioredoxin-like system. Physical, enzymatic and spectral properties of the G. duodenalis disulphide reductase are also consistent with it being a member of the thioredoxin reductase-class of disulphide reductases. Furthermore, the internal amino acid sequence of a tryptic peptide generated from the purified protein was highly homologous with thioredoxin reductases from other sources. This is the first report of a disulphide reductase to be purified from the anaerobic protozoa and explains the so called "glutathione-induced thiol-reductase activity' previously observed in G. duodenalis.
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PMID:A thioredoxin reductase-class of disulphide reductase in the protozoan parasite Giardia duodenalis. 902 54

Mammalian selenocysteine-containing thioredoxin reductase (TR) isolated from HeLa cells and from human lung adenocarcinoma cells was separated into two major enzyme species by heparin-agarose affinity chromatography. The low-affinity enzyme forms that were not retained on heparin agarose showed strong crossreactivity in immunoblot assays with anti-rat liver TR polyclonal antibodies, whereas the high-affinity enzyme forms that were retained by the heparin column were not detected. Both low and high heparin-affinity enzyme forms contained FAD, were indistinguishable on SDS/PAGE analysis, and exhibited similar catalytic activities in the NADPH-dependent DTNB [5,5'-dithiobis(2-nitrobenzoate)] assay. The C-terminal amino acid sequences of 75Se-labeled tryptic peptides from lung adenocarcinoma low- and high heparin-affinity enzyme forms were identical to the predicted C-terminal sequence of human placental TR. These two determined peptide sequences were -Ser-Gly-Ala-Ser-Ile-Leu-Gln-Ala-Gly-Cys-Secys-(Gly). Occurrence of the Se-carboxymethyl derivative of radioactive selenocysteine in the position corresponding to TGA in the gene confirmed that UGA is translated as selenocysteine. The presence of cysteine followed by a reactive selenocysteine residue in this C-terminal region of the protein may explain some of the unusual properties of the mammalian TRs.
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PMID:Heparin-binding properties of selenium-containing thioredoxin reductase from HeLa cells and human lung adenocarcinoma cells. 917 83


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