Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase

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The dithiol protein tryparedoxin is a component of the unique trypanothione/trypanothione reductase metabolism of trypanosomatids and is involved in the parasite synthesis of deoxyribonucleotides and the detoxication of hydroperoxides. Tryparedoxin is a highly abundant protein in all life stages of Trypanosoma brucei, the causative agent of African sleeping sickness. As shown here, its functional properties are intermediate between those of classical thioredoxins and glutaredoxins. The redox potential of T. brucei tryparedoxin of ؊249 mV was determined by protein-protein redox equilibration with Escherichia coli thioredoxin. The trypanothione/ tryparedoxin couple is probably the most significant factor determining the cytosolic redox potential of the parasites. The pK value of Cys 40 , the first thiol in the WCPPC motif, is 7.2 as derived from the thiolate absorption at 240 nm and the rate of carboxymethylation. Alteration of the active site into that of thioredoxin (CGPC) did not affect the pK value. In contrast, in the mutant with the glutaredoxin motif (CPYC) the pK dropped to <4.0. The fact that the pK value of tryparedoxin coincides with the intracellular pH of the parasite may contribute to the reactivity of tryparedoxin in thiol disulfide exchange reactions.

“…The reduced form, trypanothione, T(SH) 2 1 was prepared by NaBH 4 reduction as described (14). Dehydroascorbate was from Serva.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Reckenfelderbäumer

Krauth‐Siegel

2002

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“…Accordingly, also knock-down of TXN and TXN-Px by double strand RNA interference leads to impaired viability and peroxide resistance of T. brucei (11). Although trypanosomatids are also equipped with a typical thioredoxin (12), TXN appears to substitute for thioredoxin in ribonucleotide reduction (13), which may explain the growth arrest observed upon depletion of T(SH) 2 (3) or trypanothione reductase (1).…”

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confidence: 99%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

In Crithidia fasciculata the biosynthesis of trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBank TM accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg 2؉ -ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K m values for GSH, Mg 2؉ -ATP, and glutathionylspermidine were 407, 222, and 480 M, respectively, and the k cat was 8.7 s ؊1 for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.

“…Ribonucleotide Reductase Assay-The activity of T. brucei ribonucleotide reductase was determined from the rate of conversion of [ 3 H]GDP into [ 3 H]dGDP as described (4). The assay mixture contained in a total volume of 200 l of 50 mM HEPES, pH 7.6, 500 M GDP (including 1.25 Ci of [ 3 H]GDP), 100 M dTTP, 100 mM KCl, and 6.4 mM MgCl 2 .…”

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confidence: 99%

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

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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)...