Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

Reckenfelderbäumer, Nina; Lüdemann, Heike; Schmidt, Heide; Steverding, Dietmar; Krauth‐Siegel, R. Luise

doi:10.1074/jbc.275.11.7547

Cited by 70 publications

(74 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…At one time, thioredoxin was considered to be absent in Leishmania (see Ref. 25); however, a thioredoxin-like gene has been identified in L. major (GenBank TM /EBI accession number AAG10802) and T. brucei (43). One possible function of the interaction between a sulfurtransferase and thioredoxin could be the involvement of the enzyme in reduction and detoxification of peroxides (4).…”

Section: Discussionmentioning

confidence: 99%

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

Williams

Kelly

Mottram

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cytosolic 3-mercaptopyruvate sulfurtransferases (EC 2.8.1.2) of Leishmania major and Leishmania mexicana have been cloned, expressed as active enzymes in Escherichia coli, and characterized. The leishmanial singlecopy genes predict a sulfurtransferase that is structurally peculiar in possessing a C-terminal domain of some 70 amino acids. Homologous genes of Trypanosoma cruzi and Trypanosoma brucei encode enzymes with a similar C-terminal domain, suggesting that this feature, not known in any other sulfurtransferase, is a characteristic of trypanosomatid parasites. Short truncations of the C-terminal domain resulted in misfolded inactive proteins, demonstrating that the domain plays some key role in facilitating correct folding of the enzymes. The leishmanial recombinant enzymes exhibited high activity toward 3-mercaptopyruvate and catalyzed the transfer of sulfane sulfur to cyanide to form thiocyanate. They also used thiosulfate as a substrate and reduced thioredoxin as the accepting nucleophile, the latter being oxidized. The enzymes were expressed in all life cycle stages, and the expression level was increased under peroxide or hypo-sulfur stress. The results are consistent with the enzymes having an involvement in the synthesis of sulfur amino acids per se or iron-sulfur centers of proteins and the parasite's management of oxidative stress.

show abstract

Section: Discussionmentioning

confidence: 99%

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

Williams

Kelly

Mottram

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The corresponding Cys-69 in human thioredoxin has recently been shown to be accessible to nitrosylation (11). T. brucei thioredoxin catalyzes the reduction of insulin disulfide and the parasite ribonucleotide reductase by dithioerythritol and is a substrate of human thioredoxin reductase (8). In all known organisms, oxidized thioredoxin, Trx-S 2 , 1 is reduced by NADPH and the flavoenzyme thioredoxin reductase (12,13), whereby two different types of enzymes have evolved (14).…”

mentioning

confidence: 99%

“…Besides tryparedoxin, T. brucei possesses a single classical thioredoxin with a M r of 12,000 and the conserved WCGPC catalytic site (8). The parasite protein is unusual in having a calculated pI value of 8.5 instead of the acidic pI found for most thioredoxins and a conserved functional aspartate (Asp-26 in Escherichia coli thioredoxin) (9,10) is replaced by a tryptophan residue.…”

mentioning

confidence: 99%

“…In a total volume of 800 l of 100 mM potassium phosphate, 2 mM EDTA, pH 7.0, the assay mixture contained 130 M Western Blot Analysis-Procyclic T. brucei (cell line 449) were grown as described (8). The cells were harvested at a density of 3.5 ϫ 10 6 cells/ml and directly lysed in sample buffer containing tris(2-carboxyethyl)phosphine (Bond-Breaker, Pierce).…”

mentioning

confidence: 99%

“…Purification of Recombinant T. brucei Thioredoxin-E. coli SG13009 cells with the pQE32/trx plasmid were grown as described (8) but terrific broth medium was used. Cells from 1 liter of bacterial culture were suspended in 30 ml of buffer A (50 mM sodium phosphate, 300 mM NaCl, pH 7.2) containing 1 mM imidazol, 150 nM pepstatin, 4 nM cystatin, 20 M phenylmethylsulfonyl fluoride, 5 mg of lysozyme, and 0.5 mg of DNase I.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of ؊267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mM ؊1 cm ؊1 , there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/ trypanothione reductase system. Trypanosomatids such as Trypanosoma brucei, the causative agent of African sleeping sickness, have a unique thiol metabolism in which the ubiquitous glutathione/glutathione reductase couple is replaced by trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), and the flavoenzyme trypanothione reductase (1, 2). The parasite dithiol is much more reactive than glutathione. It is a spontaneous reductant of dehydroascorbate as well as the disulfide forms of glutathione and ovothiol (2). It reduces tryparedoxin, a 16-kDa dithiol protein with a CPPC active site motif, which distantly belongs to the thioredoxin protein family (3). The trypanothione/tryparedoxin couple is the donor of reducing equivalents for the detoxication of hydroperoxides catalyzed by a cascade of proteins that are composed of trypanothione reductase, trypanothione, tryparedoxin, and a peroxiredoxin-type tryparedoxin peroxidase. Tryparedoxin is also involved in the reduction of ribonucleotide reductase (4) and glutathione peroxidase-like parasite peroxidases (5, 6). The trypanothione metabolism is essential for the parasite. Down-regulation of trypanothione reductase in bloodstream T. brucei by more than 90% results in growth arrest and hypersensitivity toward hydrogen peroxide as well as the loss of infectivity in a mouse model (7).Besides tryparedoxin, T. brucei possesses a single classical thioredoxin with a M r of 12,000 and the conserved WCGPC catalytic site (8). The parasite protein is unusual in having a calculated pI value of 8.5 instead of the acidic pI found for most thioredoxins and a conserved functional aspartate (Asp-26 in Escherichia coli thioredoxin) (9, 10)...

show abstract

Trypanothione‐Based Redox Metabolism of Trypanosomatids

Comini

Flohé

2013

Trypanosomatid Diseases

View full text Add to dashboard Cite

Identification and Functional Characterization of Thioredoxin from Trypanosoma brucei brucei

Cited by 70 publications

References 49 publications

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

3-Mercaptopyruvate Sulfurtransferase of LeishmaniaContains an Unusual C-terminal Extension and Is Involved in Thioredoxin and Antioxidant Metabolism

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Trypanothione‐Based Redox Metabolism of Trypanosomatids

Contact Info

Product

Resources

About