Cloning and sequencing of a human thioredoxin reductase

Gasdaska, Pamela Y.; Gasdaska, John R.; Cochran, Shawn; Powis, Garth

doi:10.1016/0014-5793(95)01003-w

Cited by 190 publications

(188 citation statements)

References 46 publications

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“…The purified protein was analyzed by electrospray ionization (ESI) mass spectrometry. The recorded ESI-MS spectra showed a broad peak centered at 54,860 Da, that was 230 Da more than the mass predicted from the previously reported human TR1 sequence (24). Reduction and carboxymethylation of the enzyme increased its mass to 55,626 Da, but did not significantly narrow the mass peak.…”

Section: Heterogeneity Within Mouse Thioredoxinmentioning

confidence: 50%

Heterogeneity within Animal Thioredoxin Reductases

Sun¹,

Zappacosta²,

Factor³

et al. 2001

Journal of Biological Chemistry

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Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits. We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA. These forms differed in 5 sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR. In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-Omethyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membranebound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by ϳ30 kilobases. Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle-and cell type-specific location of animal thioredoxin reductases.

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Section: Heterogeneity Within Mouse Thioredoxinmentioning

confidence: 50%

Heterogeneity within Animal Thioredoxin Reductases

Sun¹,

Zappacosta²,

Factor³

et al. 2001

Journal of Biological Chemistry

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“…Large amounts of chaperone proteins were obtained in the soluble fraction but expression of functional LipDH was not increased (data not shown). Difficulties in the expression of the active recombinant enzyme have also been reported for human LipDH (Kim et al, 1991), for thioredoxin reductase (Gasdaska et al, 1995), a closely related protein, as well as for other components of complexes where LipDH is part of (Wynn et al, 1992;Maeng et al, 1994, Korotchkina et al, 1995. Expression of 7: brucei LipDH in E. coli resulted in minute amounts of active enzyme (Else et al, 1993).…”

Section: Overexpression Of T Cruzi Ipd In Escherichia Colimentioning

confidence: 98%

“…His456 which is the possible proton acceptor/donor during catalysis and Glu461 are provided by the second subunit of the homodimeric enzyme. They are conserved in all LipDH as well as in glutathione reductase, trypanothione reductase and human thioredoxin reductase (Gasdaska et al, 1995).…”

Section: Comparison Of T Cruzi Lipdh With Other Lipdh Thementioning

confidence: 99%

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

Schöneck

Billaut-Mulot

Numrich

et al. 1997

European Journal of Biochemistry

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This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50066. Comparison of the deduced amino acid sequence of LipDH from 7: crzizi with that of Trypanosoma brucei and man shows identities of 81 % and 50%, respectively.An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpdl of 7: cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for AsnlO which represents the N-terminus of the mature protei-n. This system allowed the synthesis of 1000 U 7: cruzi LipDH/l bacterial cell culture. The recombinant protein was purified to homogeneity by (NH,),SO,-precipitation and affinity chromatography on 5' AMP-Sepharose. The K,,, values for NAD', NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite.LipDH is present in all major developmental stages of 7: cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite.In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason 7: cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of 7: cruzi LipDH in drug activation and the crystallization of the protein.

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“…The mammalian TrxR directly reduces not only Trx from different species but also many nondisulfide substrates, such as selenite (8), lipid hydroperoxides (9), and H 2 O 2 (10). Sequencing and cloning demonstrated that cytosolic human and rat TrxR are highly similar to GR (11,12) and not to its E. coli counterpart (13). The sequence contains one active-site sequence motif-Cys 59 -Val-Asn-Val-Gly-Cys 64 -identical to the redox-active disulfide of GR (14).…”

mentioning

confidence: 99%

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.

452

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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Cloning and sequencing of a human thioredoxin reductase

Cited by 190 publications

References 46 publications

Heterogeneity within Animal Thioredoxin Reductases

Heterogeneity within Animal Thioredoxin Reductases

Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi

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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