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 complete amino acid sequence of the D-glucosaminate dehydratase (GADH) alpha-subunit from Pseudomonas fluorescens was determined by PCR using genomic DNA from P. fluorescens as a template. The alpha-subunit comprises 320 amino acids and has a molecular mass of about 33.9 kDa. The primary structure of the alpha-subunit demonstrates a high similarity to the structures of thioredoxin reductase (TrxR) from many prokaryotes, especially Pseudomonas aeruginosa (identity 85%, positive 91%), Vibrio cholerae (identity 73%, positive 85%), and Escherichia coli (identity 71%, positive 83%). The purified glucosaminate dehydratase alpha(2)-enzyme exhibited NADPH-dependent TrxR activity, while TrxR from E. coli showed pyridoxal 5'-phosphate (PLP)-dependent GADH activity. The TrxR from E. coli suggests that there are three cofactor binding sites, FAD, NADPH, and PLP in the enzyme and that TrxR catalyzes the FAD- and NADPH-dependent oxidation-reduction reaction and the PLP-dependent alpha,beta-elimination reaction.
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PMID:The D-glucosaminate dehydratase alpha-subunit from Pseudomonas fluorescens exhibits thioredoxin reductase activity. 1268 50

The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide-->NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (Km=8.5 microm, kcat=15.4 s(-1) at pH 7.4 and 25 degrees C) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redox-active Cys-Cys sequence.
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PMID:Thioredoxin reductase from the malaria mosquito Anopheles gambiae. 1462 92

From Bacillus subtilis cell extracts, ferredoxin-NADP+ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a approximately 94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with Km values of 0.57 microM vs >200 microM. Kcat values of YumC with NADPH were 22.7 s(-1) and 35.4 s(-1) in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.
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PMID:Purification and characterization of ferredoxin-NADP+ reductase encoded by Bacillus subtilis yumC. 1525 6

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

A thioredoxin reductase (TrxR) has been identified in the hyperthermophilic archaeon Sulfolobus solfataricus (Ss). This enzyme is a homodimeric flavoprotein that was previously identified as NADH oxidase in the same micro-organism ('Biotechnol. Appl. Biochem. 23 (1996) 47'). The primary structure of SsTrxR is made of 323 amino acid residues and contains two putative betaalphabeta regions for the binding of FAD, and a NADP(H) binding consensus sequence in the proximity of a CXXC motif. These findings indicate that SsTrxR is structurally related to the class II of the pyridine nucleotide-disulphide oxidoreductases family. Moreover, the enzyme exhibits a NADP(H) dependent thioredoxin reductase activity requiring the presence of FAD. Surprisingly, the reductase activity of SsTrxR is reduced in the presence of a specific inhibitor of mammalian TrxR. This finding demonstrates that the archaeal enzyme, although structurally related to eubacterial TrxR, is functionally closer to eukaryal enzymes. Experimental evidences indicate that a disulphide bridge is required for the reductase but also for the NADH oxidase activity of the enzyme. These results are further supported by the significantly reduced activities exerted by the C147A mutant. The integrity of the CXXC motif is also involved in the stability of the enzyme.
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PMID:A 35 kDa NAD(P)H oxidase previously isolated from the archaeon Sulfolobus solfataricus is instead a thioredoxin reductase. 1566 38

The thioredoxin system exists ubiquitously and participates in essential antioxidant and redox-regulation processes via a pair of conserved cysteine residues. In Mycobacterium tuberculosis, which lacks a genuine glutathione system, the thioredoxin system provides reducing equivalents inside the cell. The three-dimensional structure of thioredoxin reductase from M. tuberculosis has been determined at 3 A resolution. TLS refinement reveals a large libration axis, showing that NADPH-binding domain has large anisotropic disorder. The relative rotation of the NADPH domain with respect to the FAD domain is necessary for the thioredoxin reduction cycle, as it brings the spatially distant reacting sites close together. Normal-mode analysis carried out based on the elastic network model shows that the motion required to bring about the functional conformational change can be accounted for by motion along one single mode. TLS refinement and normal-mode analysis thus enhance our understanding of the associated conformational changes.
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PMID:Conformational flexibility of Mycobacterium tuberculosis thioredoxin reductase: crystal structure and normal-mode analysis. 1630 94

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.
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PMID:Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells. 1635 62

High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.
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PMID:Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum. 1695 Jul 93

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec-->Cys) in complex with FAD and NADP(+) at a resolution of 2.8 A. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development.
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PMID:The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis. 1751 5

The crystal structures of homodimeric thioredoxin reductase (TrxR) from the gastric pathogen Helicobacter pylori in complex with NADP(+) have been determined for the oxidized and reduced form of the enzyme at 1.7 and 1.45 A resolution, respectively. The enzyme subunit is built up of FAD- and NAD(P)H-binding domains, each of which contain variants of the Rossmann fold typical of nucleotide-binding proteins. The majority of the amino-acid residues binding the cofactors FAD and NAD(P)H are conserved in the low-molecular-weight thioredoxin reductases. In the reduced species the isoalloxazine ring of FAD is bent along an axis passing through the N5 and N10 atoms with an angle of 22 degrees and the ribityl moiety adopts an unusual conformation. Well defined electron density shows the position of the whole NADP(+) molecule with a binding mode similar to that observed in the Escherichia coli TrxR-thioredoxin complex, although the orientation of the NAD(P)H-binding domain is different. Rigid-body rotation of this domain to the orientation observed in the E. coli TrxR-thioredoxin complex positions NADP(+) adjacent to the FAD molecule, suitable for electron transfer, without any readjustments of the conformation of NADP(+). A comparison of the binding surfaces of thioredoxin and thioredoxin reductases from H. pylori and E. coli shows that the overall surface charge distribution in these proteins is conserved and that residue substitutions that change the shape of the binding surface may account for the species-specific recognition of thioredoxin by TrxR.
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PMID:High-resolution structures of oxidized and reduced thioredoxin reductase from Helicobacter pylori. 1758 74


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