Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Abstract

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)...

Glyoxalase II of African Trypanosomes Is Trypanothione-dependent

“…In contrast, a 10-fold excess of reduced trypanothione over methylglyoxal leads to formation of mono-(lactoyl)trypanothione. Considering a cellular concentration of trypanothione of Ն350 M in bloodstream T. brucei (44) and a methylglyoxal concentration of 1-2 M reported for human blood (45) and Saccharomyces cerevisiae (46), the monothioester of trypanothione is probably the main physiological substrate of the parasite enzyme.…”

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

“…4). The two glycines together with the Thr of the TVP motif and the Tyr in the active site (corresponding to Gly 76 , Gly 77 , Thr 64 , and Tyr 23 in T. brucei Grx1) have been reported to form a binding groove for GSH on the protein surface (10,38,39). These residues are conserved in the trypanosomatid sequences.…”

“…In vitro, by far the most efficient parasite protein disulfide reductase proved to be Tpx. It was even more reactive than T. brucei thioredoxin, previously shown to catalyze the reduction of insulin by dithioerythritol or T(SH) 2 (63,64). For T. brucei RR, the T(SH) 2 /Tpx couple is an efficient reductant (16), and the replacement of Tpx by Grx1 resulted in 50 -80% lower RR activities.…”

Glutathionylation of Trypanosomal Thiol Redox Proteins

“…Reaction of the reduced PxIII with Gsp disulfide resulted in the selective modification of Cys 95 (Table 8). Thiolation of T. brucei Thioredoxin-T. brucei thioredoxin possesses three cysteinyl residues, namely the redox active Cys 31 -Cys 34 couple and Cys 68 in the C-terminal moiety of the protein (23,31). ESI-MS analysis of the reduced protein treated with 0.5 mM GSSG or Gsp disulfide revealed monothiolated protein species (Fig.…”

“…The enzyme catalyzes the trypanothione/tryparedoxin-dependent reduction of a variety of hydroperoxides and is essential for both the mammalian and the insect stages of T. brucei (21,22). T. brucei thioredoxin catalyzes reactions typical for thioredoxins such as the reduction of ribonucleotide reductase and insulin disulfide (23). The parasite thioredoxin is probably kept reduced by the spontaneous reaction with trypanothione, because the completely sequenced genomes of T. brucei as well as T. cruzi and L. major do not reveal any gene for a thioredoxin reductase (24).…”