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
            <i>Escherichia coli</i>

Arscott, L. David; Gromer, Stephan; Schirmer, R. Heiner; Becker, Katja; Williams, Charles H.

doi:10.1073/pnas.94.8.3621

Cited by 224 publications

(248 citation statements)

References 43 publications

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“…Absorption ratios A 280 nm / A 463 nm as well as extinction coefficients of the oxidized enzyme species at 463 nm were determined from spectral analyses (wild type hTrxR: e = 11.3 mM À1 cm À1 [2,3], hTrxR-16 K29R : e = 11.74 mM À1 cm À1 , hTrxRD16 K29R,H108Y : e = 13.87 mM À1 cm À1 , hTrxRD16 K29R,H108Y,A119N,V478E : e = 15.22 mM À1 cm À1 ). Oxidised hTrxR-16 had a typical flavoprotein spectrum [28] with peak absorption at 463 nm ( Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

et al. 2006

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The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.

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Section: Resultsmentioning

confidence: 99%

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

et al. 2006

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“…It has been shown that both motifs are essential for enzymatic activity with thioredoxin as substrate (10 -12, 19). Even though it has been proposed that there exists a high degree of structural similarity between the high M r TrxRs with glutathione reductase and lipoamide dehydrogenase (10,18), the catalytic mechanism of the TrxRs appears to be much more complicated than that of the latter enzymes. Wang et al (10,11) have shown that during catalysis reducing equivalents are transferred from FAD to the N-terminal pair of cysteines and subsequently reduce the C-terminal cysteine pair by an intramolecular dithiol/disulfide exchange.…”

Section: Expression and Purification Of Pftrxr Homo-and Heterodimers-mentioning

confidence: 99%

Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

Krnajski

Gilberger

Walter

et al. 2000

Journal of Biological Chemistry

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The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin. It is responsible for the reduction of disulfide bridges in proteins such as ribonucleotide reductase and several transcription factors. Furthermore, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxRs: the high M r and the low M r proteins. The well characterized Escherichia coli TrxR represents a member of the low M r class of proteins, whereas the mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins belong to the family of high M r proteins. The primary structure of these proteins is very similar to that of glutathione reductase and lipoamide dehydrogenase. However, the high M r TrxRs possess, in addition to their redox active N-terminal pair of cysteines, a pair of cysteine residues or a selenenylsulfide motif at their C terminus. These residues have been shown to be crucial for the reduction of thioredoxin. In this study we address the question whether the active site residues of P. falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants were co-expressed in E. coli and the recombinant protein species were purified by affinity chromatography specific for the respective tags of the recombinant proteins. Co-expression of PfTrxR wild-type and mutant proteins resulted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Co-expression of the double mutant PfTrxRC88AC535A with PfTrxR wild-type generated an inactive heterodimer, which indicates that PfTrxR possesses intersubunit active sites. In addition, the data presented possibly imply a coopertive interaction between both active sites of PfTrxR.Infection with Plasmodium falciparum, the causative agent of malaria tropica, is responsible for 2-3 million deaths per year. The malaria parasite spends part of its developmental life cycle in human erythrocytes where it is challenged with enhanced oxidative stress. Therefore the parasite needs efficient anti-oxidants to protect itself against damages, such as nucleic acid modifications, lipid peroxidation, or oxidation of thiolcontaining proteins, caused by reactive oxygen species. The thioredoxin redox system, composed of the NADPH-dependent homodimeric thioredoxin reductase (TrxR) 1 and the 12-kDa protein thioredoxin (Trx) confers reduction of protein disulfides, ribonucleotide reductase being the most prominent example (1). Apart from this, thioredoxin interacts with a number of transcription factors in prokaryotic and eukaryotic cells, resulting in modified DNA binding activities and altered gene transcription (2). Another important function is the interaction of reduced thioredoxin with thioredoxin-dependent peroxidases, which detoxify reactive oxyg...

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“…However, it had a 100-fold lower k cat and a 10-fold lower K m for Trx compared with the Se-containing wild-type rat enzyme (10). The truncated protein lacking the C-terminal SeCys-Gly dipeptide was also folded and contained FAD but was inactive although titration with NADPH yielded the characteristic thiolate-flavin charge transfer complex common for GR and mammalian TrxR (1,28). In this paper, we have identified a selenenylsulfide as the active site of TrxR and propose a structure model and mechanisms for the enzyme.…”

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Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

Zhong

Arnér

Holmgren

2000

Proc. Natl. Acad. Sci. U.S.A.

447

343

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Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys 59 -Val-Asn-Val-Gly-Cys 64 , identical to the active site of GR; the other was a selenenylsulfide formed from Cys 497 -SeCys 498 and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys 497 -SeCys 498 residues of one monomer are adjacent to the Cys 59 and Cys 64 residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys 59 and Cys 64 dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys 498 replaced by Cys having a 100-fold lower kcat for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys 59 -Cys 64 was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.evolution ͉ thiols ͉ disulfide ͉ selenium ͉ enzyme structure

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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

Cited by 224 publications

References 43 publications

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

Intersubunit Interactions in Plasmodium falciparumThioredoxin Reductase

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

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