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

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

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

The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH. Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase. The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data. In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both. The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear. To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain. The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol-disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer. Reverse rotation allows reduction of the redox active disulfide by the reduced flavin. This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed.
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PMID:Mechanism and structure of thioredoxin reductase from Escherichia coli. 755 16

The DNA sequence encoding human placental thioredoxin reductase has been determined. Of the 3826 base pairs sequenced, 1650 base pairs were in an open reading frame encoding a mature protein with 495 amino acids and a calculated molecular mass of 54,171. Sequence analysis showed strong similarity to glutathione reductases and other NADPH-dependent reductases. Human thioredoxin reductase contains the redox-active cysteines in the putative FAD binding domain and has a dimer interface domain not previously seen with prokaryote and lower eukaryote thioredoxin reductases.
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PMID:Cloning and sequencing of a human thioredoxin reductase. 758 32

Human thioredoxin reductase is a dimeric enzyme that catalyzes reduction of the disulfide in oxidized thioredoxin by a mechanism involving transfer of electrons from NADPH via FAD to a redox-active disulfide. 1-Chloro-2,4-dinitrobenzene (DNCB) is an alkylating agent used for depleting intracellular GSH and also showing distinct immunomodulatory properties. We have discovered that low concentrations of DNCB completely inactivated human or bovine thioredoxin reductase, with a second order rate constant in excess of 200 M-1 s-1, which is almost 10,000-fold faster than alkylation of GSH. Total inactivation of 50 nM reduced thioredoxin reductase was obtained by 100 microM DNCB after 5 reductase was obtained by 100 microM DNCB after 5 min of incubation at 20 degrees C also in the presence of 1 mM GSH. The inhibition occurred with enzyme only in the presence of NADPH and persisted after removal of DNCB, suggesting alkylation of the active site nascent thiols as the mechanism of inactivation. Thioredoxin reductase modified by DNCB lacked reducing activity with oxidized thioredoxin, 5,5'-dithiobis-(2-nitrobenzoic acid), or sodium selenite. However, the DNCB-modified enzyme oxidized NADPH at a rate of 4.7 nmol/min/nmol of enzyme in the presence of atmospheric oxygen. This activity was not dependent on the presence of DNCB in solution and constituted a 34-fold increase of the inherent low NADPH oxidase activity of the native enzyme. DNCB is a specific inhibitor of mammalian thioredoxin reductase, which reacted 100-fold faster than glutathione reductase. The inactivation of the disulfide reducing activity of thioredoxin reductase and thioredoxin with a concomitant large increase of the NADPH oxidase activity producing reactive oxygen intermediates may mediate effects of DNCB on cells in vivo.
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PMID:1-Chloro-2,4-dinitrobenzene is an irreversible inhibitor of human thioredoxin reductase. Loss of thioredoxin disulfide reductase activity is accompanied by a large increase in NADPH oxidase activity. 787 79

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

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.
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PMID:Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. 865 Feb 34


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