A Second Class of Peroxidases Linked to the Trypanothione Metabolism

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.

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confidence: 99%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

“…9). T. brucei thioredoxin can replace tryparedoxin in the reactions catalyzed by C. fasciculata tryparedoxin peroxidase I as well as by the T. brucei glutathione peroxidase-type enzyme as shown recently (6). In both cases, thioredoxin is less efficient than tryparedoxin.…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 98%

“…brucei thioredoxin also provides electrons for the parasite detoxication of hydroperoxides. Two different types of peroxidases, 2 Cys-peroxiredoxins and glutathione peroxidase-related enzymes, occur in trypanosomatids (2,5,6,28,40). Both types of enzymes obtain their reducing equivalents from NADPH via a unique sequence of reactions involving trypanothione reductase, trypanothione, and tryparedoxin (Fig.…”

Section: Reduction Of T Brucei Thioredoxin By Trypanothione and Othermentioning

confidence: 99%

“…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.…”

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confidence: 99%

See 1 more Smart Citation

Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei

Schmidt

Krauth‐Siegel

2003

Self 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)...

“…These are homologues of the selenoprotein glutathione peroxidase (GPX), with the active site selenocysteine being replaced by a cysteine residue. However, with the exception of the T. cruzi GPX II (20), these enzymes use TryX, rather than GSH as their preferred reductant (14,19).In mammalian cells, another class of enzymes that are im-* This work was supported by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

Leishmania major Elongation Factor 1B Complex Has Trypanothione S-Transferase and Peroxidase Activity

Vickers

Wyllie

Fairlamb

2004

In the Trypanosomatidae, trypanothione has subsumed many of the roles of glutathione in defense against chemical and oxidant stress. Crithidia fasciculata lacks glutathione S-transferase, but contains an unusual trypanothione S-transferase activity that is associated with eukaryotic translation elongation factor 1B (eEF1B). Here we describe the cloning, expression, and reconstitution of the purified ␣, ␤, and ␥ subunits of eEF1B from Leishmania major. Individual subunits lacked trypanothione S-transferase activity. Only eEF1B, formed by reconstitution or co-expression of the three subunits, was able to conjugate a variety of electrophilic substrates to trypanothione or glutathionylspermidine, but not glutathione. In contrast to the C. fasciculata eEF1B, the L. major enzyme also displayed peroxidase activity against a variety of organic hydroperoxides. The enzyme showed no activity with hydrogen peroxide and greatest activity with linoleic acid hydroperoxide (1 unit mg ؊1 ). Kinetic studies suggest a ternary complex mechanism, with K m values of 140 M for trypanothione and 7.4 mM for cumene hydroperoxide and k cat ‫؍‬ 25 s ؊1 . Immunofluorescence studies indicate that the enzyme may be localized to the surface of the endoplasmic reticulum. These results suggest that, in addition to its role in protein synthesis, the Leishmania eEF1B may help protect the parasite from lipid peroxidation.Parasitic protozoa of the family trypanosomatidae cause disease and death throughout the tropical and subtropical world. Trypanosoma brucei infections are estimated to cause ϳ400,000 cases of sleeping sickness per year; Trypanosoma cruzi, the cause of Chagas' disease, chronically infects ϳ17 million people and Leishmania spp. are thought to cause 2 million cases of leishmaniasis per year. 1 Current chemotherapies for these diseases are, on the whole, ineffective and toxic (2, 3), whereas effective vaccines may never be developed. The development of effective treatments for these infections is therefore an urgent necessity.New anti-parasitic drugs can be developed from inhibitors of biochemical pathways that are essential for parasite survival but absent from the host. One such target is the thiol metabolism of trypanosomatids. Uniquely, this is dependent upon trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine or T[SH] 2 ) 2 (4), whereas their human hosts use glutathione ((␥-L-glutamyl-L-cysteinylglycine or GSH). Trypanothione is involved in protective processes, defending against oxidative stress through peroxidase systems (5), against reactive aldehydes through the glyoxalase system (6, 7), and against toxic xenobiotics through the trypanothione S-transferase (TST) (8). All these processes depend upon trypanothione being maintained in its dithiol form by trypanothione reductase. This NADPH-dependent flavoenzyme is essential to the trypanosomatids (9, 10) as, in these organisms, it is the only known route for the transfer of reducing equivalents from NADPH to low molecular mass thiols. In addition, because these parasites lack an...