Catalytic Properties, Thiol p K Value, and Redox Potential of Trypanosoma brucei Tryparedoxin

Reckenfelderbäumer, Nina; Krauth‐Siegel, R. Luise

doi:10.1074/jbc.m112115200

Cited by 51 publications

(58 citation statements)

References 37 publications

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“…2b and 5) and similar biophysical properties (35,36) of TbTryX as compared with the C. fasciculata tryparedoxins suggest that the three-dimensional structures should be similar. An overlap of CfTryX1, CfTryX2, and TbTryX highlights the structural homology of tryparedoxins (Fig.…”

Section: Structure Of Tbtryx and A Model For The Interaction With Tryp-mentioning

confidence: 99%

Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

Alphey¹,

Gabrielsen²,

Micossi

et al. 2003

Journal of Biological Chemistry

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Tryparedoxin (TryX) is a member of the thioredoxin (TrX) fold family involved in the regulation of oxidative stress in parasitic trypanosomatids. Like TrX, TryX carries a characteristic Trp-Cys-Xaa-Xaa-Cys motif, which positions a redox-active disulfide underneath a tryptophan lid. We report the structure of a Crithidia fasciculata tryparedoxin isoform (CfTryX2) in two crystal forms and compare them with structures determined previously. Efforts to chemically generate crystals of reduced TryX1 were unsuccessful, and we carried out a novel experiment to break the redox-active disulfide, formed between Cys-40 and Cys-43, utilizing the intense x-radiation from a third generation synchrotron undulator beamline. A time course study of the S-S bond cleavage is reported with the structure of a TryX1 C43A mutant as the control. When freed from the constraints of a disulfide link to Cys-43, Cys-40 pivots to become slightly more solventaccessible. In addition, we have determined the structure of Trypanosoma brucei TryX, which, influenced by the molecular packing in the crystal lattice, displays a significantly different orientation of the active site tryptophan lid. This structural change may be of functional significance when TryX interacts with tryparedoxin peroxidase, the final protein in the trypanothione-dependent peroxidase pathway. Comparisons with chloroplast TrX and its substrate fructose 1,6-bisphosphate phosphatase suggest that this movement may represent a general feature of redox regulation in the trypanothione and thioredoxin peroxidase pathways.

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Section: Structure Of Tbtryx and A Model For The Interaction With Tryp-mentioning

confidence: 99%

Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

Alphey¹,

Gabrielsen²,

Micossi

et al. 2003

Journal of Biological Chemistry

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“…A corresponding behavior has been reported for E. coli thioredoxin by Mössner et al (38), whereby Kallis and Holmgren (37) found at pH 5.7 a k app of 44.2 M Ϫ1 s Ϫ1 . Also the reaction rates of T. brucei tryparedoxin and DsbA with iodoacetic acid did not drop to zero at low pH values (19,21). The maximum reaction rate of T. brucei thioredoxin with iodoacetamide is 60 M Ϫ1 s Ϫ1 at pH 8.5.…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 79%

“…Most probably this reflects the pK value of Cys-31 but alkylation of Cys-34 cannot be excluded because the thiolate titration experiments indicate that in T. brucei thioredoxin both cysteines have very similar pK values. Carboxamidomethylation of the protein did not show a second inflection point at higher pH values in contrast to E. coli thioredoxin (37,38) and T. brucei tryparedoxin (21).…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 99%

“…A titration curve with a single transition at pH 7.4 was also obtained with several active site mutants of E. coli thioredoxin in contrast to wild type E. coli thioredoxin (38) and T. brucei tryparedoxin (21) where the pH dependence of the alkylation rate results in a second inflection point. In E. coli thioredoxin, this was attributed to an increased reactivity of the nucleophilic cysteine at higher pH values because of the titration of another residue influencing its reactivity (38).…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 99%

“…Determination of the Redox Potential-The redox potential of T. brucei thioredoxin was determined by direct protein-protein redox equilibration (20,21). Different concentrations of reduced and oxidized T. brucei and E. coli thioredoxin in 100 l of 100 mM potassium phosphate, pH 7.0, were allowed to equilibrate at 25°C in closed 1.5-ml reaction vessels purged with argon.…”

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Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Journal of Biological Chemistry

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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)...

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Schwefel und Selen: Bedeutung der Oxidationsstufe für Struktur und Funktion von Proteinen

Jacob

Giles

et al. 2003

Angewandte Chemie

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Schwefel und Selen kommen in Proteinen als Bestandteile der Aminosäuren Cystein, Methionin, Selenocystein und Selenomethionin vor, deren Ausnahmestellung in neueren Arbeiten unterstrichen wird. Ihre Redoxeigenschaften unter physiologischen Bedingungen ermöglichen eine erstaunliche Vielfalt posttranslationaler Proteinmodifikationen, metallfreier Redoxreaktionen und ungewöhnlicher Chalkogenredoxzustände. Wie keine andere Aminosäure kann das “Redox‐Chamäleon” Cystein an so unterschiedlichen Redoxreaktionen wie Atom‐, Elektronen‐ und Hydridtransfer sowie Radikal‐ und Austauschreaktionen beteiligt sein. Es kommt im menschlichen Körper in zahlreichen Oxidationsstufen vor, von denen jede mit charakteristischen chemischen (z. B. Redox‐ und Metallbindungs‐) und biologischen Eigenschaften verknüpft ist. Durch die Position des Selens im Periodensystem der Elemente zwischen Metallen und Nichtmetallen werden Selenoproteine zu idealen Katalysatoren einer Vielzahl biologischer Redoxumwandlungen, sodass die chalkogenhaltigen Aminosäuren Cystein, Methionin, Selenocystein und Selenomethionin und ihre einzigartige Biochemie Gegenstand eines vielversprechenden Forschungsgebietes sind.

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Catalytic Properties, Thiol p K Value, and Redox Potential of Trypanosoma brucei Tryparedoxin

Cited by 51 publications

References 37 publications

Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

Tryparedoxins from Crithidia fasciculata and Trypanosoma brucei

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schwefel und Selen: Bedeutung der Oxidationsstufe für Struktur und Funktion von Proteinen

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