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)

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

A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.
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PMID:Rat liver thioredoxin and thioredoxin reductase: purification and characterization. 715 51

Thioredoxin exists in all organisms and is responsible for the hydrogen transfer to important enzymes for ribonucleotide reduction and the reduction of methionine sulphoxide and sulphate. Thioredoxins have also been shown to regulate enzyme activity in plants and are also involved in the regulation of transcription factors and several other regulatory activities. Thioredoxin is reduced by the flavoenzyme thioredoxin reductase using NADPH. We have now determined the first structure of a eukaryotic thioredoxin reductase, from the plant Arabidopsis thaliana, at 2.5 A resolution. The dimeric A. thaliana thioredoxin reductase is structurally similar to that of the Escherichia coli enzyme, and most differences occur in the loops. Because the plant and E. coli enzymes have the same architecture, with the same dimeric structure and the same position of the redox active disulphide bond, a similar mechanism that involves very large domain rotations is likely for the two enzymes. The subunit is divided into two domains, one that binds FAD and one that binds NADPH. The relative positions of the domains in A. thaliana thioredoxin reductase differ from those of the E. coli reductase. When the FAD domains are superimposed, the NADPH domain of A. thaliana thioredoxin reductase must be rotated by 8 degrees to superimpose on the corresponding domain of the E. coli enzyme. The domain rotation we now observe is much smaller than necessary for the thioredoxin reduction cycle.
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PMID:Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution. 900 Jun 29

Thioredoxin (Trx) is a small ubiquitous dithiol protein which together with the FAD-containing enzyme thioredoxin reductase (TR) and NADPH (the Trx system) is a hydrogen donor for ribonucleotide reductase essential for DNA synthesis and a general protein disulfide reductase involved in redox regulation. Selenite, selenodiglutathione (GS-Se-SG) and selenocystine are efficiently reduced by thioredoxins and also directly by NADPH and mammalian TR but not by the E. coli enzyme. Incubation of selenite or GS-Se-SG with the Trx system or with mammalian TR results in a rapid formation of selenide, which by redox cycling with oxygen may cause a large non-stoichiometric oxidation of NADPH. Selenocystine is efficiently reduced into two molecules of the selenol amino acid selenocysteine by mammalian TR with a K(m)-value (6 mumol.L-1) and a high turnover number (kappa cat 3200 min-1) almost identical to the natural substrate Trx-S2. TR also directly reduces lipid hydroperoxides and this peroxidase reaction is strongly stimulated by the presence of catalytic amounts of free selenocysteine. Glutaredoxin (Grx) which catalyzes GSH-dependent disulfide reduction also via a redox-active disulfide and Trx are both efficient electron donors to the human plasma glutathione peroxidase providing a mechanism by which human plasma glutathione peroxidase may reduce hydroperoxides in an environment almost free from glutathione. Selenate is reduced by Grx and Trx in the presence of GSH. The DNA-binding of the transcription factor AP-1 is strongly inhibited by GS-Se-SG and selenite. Furthermore, selenide formed by TR-mediated reduction of selenite and GS-Se-SG inhibits lipoxygenase and changes the electron spin resonance spectrum of the active site iron. Mammalian TR with two subunits of 57 kDa has recently been cloned and shown to be homologous to glutathione reductase. The rat enzyme contains a selenocysteine residue in a unique Cterminal position and a conserved SECIS sequence directing insertion of the selenocysteine. The discovery of selenocysteine in mammalian TR may explain the broad substrate specificity of the enzyme and the requirement of selenium for cell proliferation.
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PMID:Selenium and the thioredoxin and glutaredoxin systems. 931 20

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.
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PMID:Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin. 1137 Oct 7

Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP(+) has been determined to 3.0-A resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.
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PMID:Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme. 1148 39

Thioredoxin reductases catalyse the reduction of thioredoxin disulfide and some other oxidised cell constituents. They are homodimeric proteins containing one FAD and accepting one NADPH per subunit as essential cofactors. Some of these reductases contain a selenocysteine at the C terminus. Based on the X-ray structure of rat thioredoxin reductase, homology models of human thioredoxin reductase were created and subsequently docked to thioredoxin to model the active complex. The formation of a new type of a catalytic triad between selenocysteine, histidine and a glutamate could be detected in the protein structure. By means of DFT (B3LYP, lacv3p**) calculations, we could show that the formation of such a triad is essential to support the proton transfer from selenol to a histidine to stabilise a selenolate anion, which is able to interact with the disulfide of thioredoxin and catalyses the reductive disulfide opening. Whereas a simple proton transfer from selenocysteine to histidine is thermodynamically disfavoured by some 18 kcal mol(-1), it becomes favoured when the carboxylic acid group of a glutamate stabilises the formed imidazole cation. An identical process with a cysteine instead of selenocysteine will require 4 kcal mol(-1) more energy, which corresponds to a calculated equilibrium shift of approximately 1000:1 or a 10(3) rate acceleration: a value close to the experimental one of about 10(2) times. These results give new insights into the catalytic mechanism of thioredoxin reductase and, for the first time, explain the advantage of the incorporation of a selenocysteine instead of a cysteine residue in a protein.
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PMID:The functional role of selenocysteine (Sec) in the catalysis mechanism of large thioredoxin reductases: proposition of a swapping catalytic triad including a Sec-His-Glu state. 1565 Oct 42

Thioredoxin reductases (TrxRs) are flavin-containing dithioloxidoreductases that couple reduction equivalents from the soluble NAD(P)H pool to the soluble protein thioredoxin (Trx). Previous crystallographic studies of the Escherichia coli enzyme ( ecTrxR) have shown that low molecular weight TrxRs can adopt two distinct conformations: the first (FO) is required for the oxidation of the flavin cofactor and the generation of reduced Trx; the second (FR) is adopted for the reduction of the flavin by NAD(P)H. Here, protein electrochemistry has been used to interrogate the equilibrium between the oxidized and reduced conformations of the ecTrxR and a novel, low molecular weight TrxR from the thermophilic archaeon Thermoplasma acidophilum ( taTrxR) that is characterized structurally and biochemically in the accompanying paper [Hernandez et al. (2008) Biochemistry 47, 9728-9737]. A reversible electrochemical response is observed that reveals a dynamic behavior dependent upon the temperature of the experiment. At low temperatures (283 K) a broad, quasi-reversible electrochemical envelope is observed centered at a value of approximately -300 mV and displaying a peak width of over 150 mV. The voltammetric response sharpens dramatically as the temperature increases, becoming much more reversible (as determined by peak separation and peak width). The overall potential and shape of the voltammetric data indicate that the flavin (FAD/FADH 2) and disulfide/dithiol couples are very close in thermodynamic potentials, and the data are interpreted in terms of the model of two-state conformational change between flavin reducing (FR) and flavin oxidizing (FO) states, where the difference in potential for the flavin and disulfide cofactors must be within 40 mV of one another. In this model, the low temperature peak broadening is interpreted as an indication of a heterogeneous population of TrxR conformations that exist at low temperature; at higher temperatures, FO and FR conformers can rapidly interconvert, and voltammetry reports upon an average potential of the conformations.
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PMID:Direct electrochemical analyses of a thermophilic thioredoxin reductase: interplay between conformational change and redox chemistry. 1871 94

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.
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PMID:Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni. 2163 Jun 72

Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.
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PMID:Fragment-Based Discovery of a Regulatory Site in Thioredoxin Glutathione Reductase Acting as "Doorstop" for NADPH Entry. 2980 May 15


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