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Query: UNIPROT:P30044 (antioxidant enzyme)
 8,037 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.
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PMID:Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis. 776 8

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

The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 microM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 microM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.
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PMID:Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis: comparison of metronidazole-resistant and sensitive strains. 795 98

A 25-kDa antioxidant enzyme that provides protection against oxidation systems capable of generating reactive oxygen and sulfur species has previously been identified. The nature of the oxidant eliminated by, and the physiological source of reducing equivalents for, this enzyme, however, were not known. The 25-kDa enzyme is now shown to be a peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of hydrogens provided by thioredoxin, thioredoxin reductase, and NADPH. This protein is the first peroxidase to be identified that uses thioredoxin as the immediate hydrogen donor and is thus named thioredoxin peroxidase (TPx). TPx exists as a dimer of identical 25-kDa subunits that contain 2 cysteine residues, Cys47 and Cys170. Cys47-SH appears to be the site of oxidation by peroxides, and the oxidized Cys47 probably reacts with Cys170-SH of the other subunit to form an intermolecular disulfide. Mutant TPx proteins lacking either Cys47 or Cys170, therefore, do not exhibit thioredoxin-coupled peroxidase activity. The TPx disulfide is specifically reduced by thioredoxin, but can also be reduced (less effectively) by a small molecular size thiol. The Saccharomyces cerevisiae thioredoxin reductase gene was also cloned and sequenced, and the deduced amino sequence was shown to be 51% identical with that of the Escherichia coli enzyme.
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PMID:Thioredoxin-dependent peroxide reductase from yeast. 796 86

Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit. It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis. Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound. Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed. It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme. When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound. These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis.
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PMID:Enzyme-monitored turnover of Escherichia coli thioredoxin reductase: insights for catalysis. 866 60

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.
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PMID:The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli. 910 27

Thioredoxin reductase from Escherichia coli is a dimeric enzyme containing one FAD and one redox-active disulfide per monomer and catalyzes the transfer of electrons from NADPH to thioredoxin, which subsequently performs several important cellular functions. To overcome problems with site-directed mutagenesis and low expression, the thioredoxin reductase gene was adapted for use in the plasmid vector pSL350 (Brosius, J., Methods Enzymol. 216, 469-483, 1992), which is designed both for protein expression and for production of single-stranded template DNA for mutagenesis, and examined expression of wild-type thioredoxin reductase under different growth conditions. In the absence of IPTG inducer, expression of thioredoxin reductase in saturated cultures accounts for 19% of the soluble protein, and with 1 mM IPTG expression increases to 61%. Some of the thioredoxin reductase is expressed as apoenzyme with the amount of apoenzyme increasing at higher IPTG concentrations, accounting for as high as 68% of the total thioredoxin reductase expressed. The apoenzyme in cell extracts is activated rapidly by addition of FAD, indicating correct folding of the enzyme in the absence of cofactor. Purification of wild-type thioredoxin reductase from the new system yielded 189 mg of enzyme from a 300-ml uninduced culture. The new plasmid was also used to generate an N155Y mutant which is purified and partially characterized.
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PMID:Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation. 912 9

4-Hydroxynonenal, a product of oxidative degradation of unsaturated lipids, is an endogenous reactive alpha,beta-unsaturated aldehyde with numerous biological activities. 4-Hydroxynonenal rapidly inactivated glutathione reductase in an NADPH-dependent reaction. Inactivation appears to involve the initial formation of an enzyme-inactivator complex, K(D) = 0.5 microM, followed by the inactivation reaction, k = 1.3 x 10(-2) min(-1). alpha,beta-Unsaturated aldehydes such as acrolein, crotonaldehyde, and cinnamaldehyde also inactivated glutathione reductase, although rates varied widely. Inactivation of glutathione reductase by alpha,beta-unsaturated aldehydes was followed by slower NADPH-independent reactions that led to formation of nonfluorescent cross-linked products, accompanied by loss of lysine and histidine residues. Other reactive endogenous aldehydes such as methylglyoxal, 3-deoxyglucosone, and xylosone inactivated glutathione reductase by an NADPH-independent mechanism, with methylglyoxal being the most reactive. However, 2-oxoaldehydes were much less effective than 4-hydroxynonenal. Inactivation of glutathione reductase by these 2-oxoaldehydes was followed by slower reactions that led to the formation of fluorescent cross-linked products over a period of several weeks. These changes were accompanied by loss of arginine residues. Thus, the sequence of events is different for inactivation and modification of glutathione reductase by alpha,beta-unsaturated aldehydes compared with 2-oxoaldehydes with respect to kinetics, NADPH requirements, fluorescence changes, and loss of amino acid residues. The ability of 4-hydroxynonenal at low concentrations to inactivate glutathione reductase, a central antioxidant enzyme, suggests that oxidative degradation of unsaturated lipids may initiate a positive feedback loop that enhances the potential for oxidative damage.
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PMID:Inactivation of glutathione reductase by 4-hydroxynonenal and other endogenous aldehydes. 917 18

Human thioredoxin reductase was recently shown to contain a TGA encoded selenocysteine residue at the penultimate position of its amino acid chain. Depending on the availability of selenium during biosynthesis, an authentic selenocysteine-containing or a selenium-free enzyme truncated at the penultimate position is expected to be formed. Correspondingly, the enzymatic activity should be altered by selenium restriction, if the selenocysteine residue is functionally important. In order to check the catalytic role of the selenocysteine residue, four different human cell lines were grown in selenium deficient media or with adequate selenium supplementation (40 nM sodium selenite) and thioredoxin reductase activity was measured as NADPH-dependent DTNB reduction or thioredoxin-mediated insulin reduction. Thioredoxin reductase activities, like glutathione peroxidase activities, were consistently higher in selenium supplemented cells, whereas glutathione reductase activity was not affected by the selenium. The dose-response was similar for thioredoxin reductase and glutathione peroxidase, but the recovery of glutathione peroxidase activity upon selenium supplementation was faster than with thioredoxin reductase. Also the increase of glutathione peroxidase activities was substantially higher than that of thioredoxin reductase (400-1200% versus a maximum of 250%). These observations clearly indicate a catalytic role of the selenocysteine residue in the thioredoxin reductase, but suggest either the existence of a selenium-unresponsive isoenzyme or a residual disulfide reductase activity in the selenium-free truncated protein made under conditions of selenium deficiency.
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PMID:Evidence for a functional relevance of the selenocysteine residue in mammalian thioredoxin reductase. 928 5

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.
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PMID:Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation. 933 41


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