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In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3-7-fold higher in the parasite form infecting mammals (50-100 microM) than in the form hosted by the tsetse fly (7-34 microM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 muM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2-3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.

Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a "resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases.

Tryparedoxins are components of the hydroperoxide detoxification cascades of Kinetoplastida, where they mediate electron transfer between trypanothione and a peroxiredoxin, which reduces hydroperoxides and possibly peroxynitrite. Tryparedoxins may also be involved in DNA synthesis, by their capacity to reduce ribonucleotide reductase. Here we report on the isolation of two tryparedoxin genes from Leishmania infantum, Li7XN1 and LiTXN2, which share the same genetic locus. These genes are both single copy and code for two active tryparedoxin enzymes, LiTXN1 and LiTXN2, with different biochemical and biological features. LiTXN1 is located to the cytosol and is upregulated in the infectious forms of the parasite, strongly suggesting that it might play an important role during infection. LiTXN2 is the first mitochondrial tryparedoxin described in Kinetoplastida. Biochemical assays performed on the purified recombinant proteins have shown that LiTXN1 preferentially reduces the cytosolic L. infantum peroxiredoxins, LicTXNPx1 and LicTXNPx2, whereas LiTXN2 has a higher specific activity for a mitochondrial peroxiredoxin, LimTXNPx. Kinetically, the two tryparedoxins follow a ping-pong mechanism and show no saturation. We suggest that LiTXN1 and LiTXN2 are part of two distinct antioxidant machineries, one cytosolic, the other mitochondrial, that complement each other to ensure effective defence from several sources of oxidants throughout the development of L. infantum.

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of (15)N,(13)C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix alpha1 with the redox-active Cys 43 and helix alpha2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N(1),N(8)-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional (1)H,(15)N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.

Tryparedoxin peroxidases (TXNPx) catalyze hydroperoxide reduction by tryparedoxin (TXN) by an enzyme substitution mechanism presumed to involve three catalytic intermediates: (i) a transient oxidation state having C52 oxidized to a sulfenic acid, (ii) the stable oxidized form with C52 disulfide-bound to C173', and (iii) a semi-reduced intermediate with C40 of TXN disulfide-linked to C173' from which the ground state enzyme is regenerated by thiol/disulfide reshuffling. This kinetically unstable form was mimmicked by a dead-end intermediate generated by cooxidation of TXNPx of Trypanosoma brucei brucei with an inhibitory mutein of TXN in which C43 was replaced by serine (TbTXNC43S). Cleavage of the isolated dead-end intermediate by trypsin plus chymotrypsin yielded a fragment that complied in size with the TbTXNC43S sequence 36 to 44 disulfide-linked to the TbTXNPx sequence 169 to 177. The presumed nature of the proteolytic fragment was confirmed by MS/MS sequencing. The results provide direct chemical evidence for the assumption that the reductive part of the catalysis is initiated by an attack of the substrate's solvent-exposed C40 on C173' of the oxidized peroxidase and, thus, confirm the hypothesis on the interaction of 2-Cys-peroxiredoxins with their proteinaceous substrates.

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidases by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3D-structures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the 1N-glutathionyl residue of trypanothione, as evidenced by the structure of 1N-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXN-substrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the 1N-glutathionyl-spermidine binding in the CfTXN2H6-Cys44Ser structure.

Tryparedoxins (TXN) are thioredoxin-related proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothione-dependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, 40WCPPCR45, of Crithidia fasciculata was mutated by site-directed mutagenesis and eight corresponding muteins were expressed in E. coli as terminally His-tagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarilythe reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin-like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin-like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.

The thioredoxin-related protein tryparedoxin I from Crithidia fasciculata has been crystallized using PEG 4000 as a precipitant. The enzyme forms long needle-shaped crystals which diffract to at least 1.7 A. A native data set has been collected at the DESY synchrotron from a flash-frozen crystal at 90 K to 1.7 A resolution. The data set shows that the crystals belong to the orthorhombic space group P212121 and have unit-cell parameters a = 37.94, b = 51. 39, c = 71.46 A. Tryparedoxin I is involved in a trypanothione-dependent peroxide metabolic pathway specific for trypanosomatids and may therefore be a suitable candidate for the design of drugs for the specific treatment of a variety of important tropical diseases caused by these parasites.

Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related proteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic parameters similar to those of the authentic enzyme in the trypanothione/peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as potential trypanocidal agents.

Tryparedoxin has recently been discovered as a constituent of the trypanosomal peroxidase system catalysing the reduction of a peroxiredoxin-type peroxidase by trypanothione [Nogoceke et al. (1997) Biol. Chem. 378, 827-836] and has attracted interest as a potential molecular target for the development of trypanocidal agents. Here we describe the first isolation of a novel gene from Crithidia fasciculata encoding a different tryparedoxin designated tryparedoxin II. The deduced amino acid sequence of tryparedoxin II (accession number AF055986) differs substantially from the partial sequence reported for the tryparedoxin described previously and now renamed tryparedoxin I. It shares the sequence motif Vx3FSAxWCPPCR shown to represent the catalytic site in tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918] with mouse nucleoredoxin (accession number X92750), and a thioredoxin-like gene product of Caenorhabditis elegans (accession number U23511). Depending on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger than the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagged tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (KM peroxiredoxin = 4.2 microM, KM trypanothione = 33 microM, Vmax/[E] = 952 min(-1)) similar to those reported for tryparedoxin I [Gommel et al. (1997) Eur. J. Biochem. 248, 913-918]. The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.

Tryparedoxin, a thioredoxin-related protein from Crithidia fasciculata with a molecular mass of 16 kDa catalyses the reduction of a peroxiredoxin-type peroxidase, Cf21, at the expense of trypanothione [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. E. (1997) Biol. Chem. Hoppe-Seyler 378, 827-836]. The kinetic analysis of tryparedoxin revealed an enzyme substitution mechanism. The corresponding molecular event was elucidated to be a reversible oxidoreduction of the disulfide bridge in the thioredoxin-related motif WCPPC. The amino-proximal cysteine residue of this active site was more reactive in S-alkylation experiments than the distal residue. The natural substrates of tryparedoxin, trypanothione and Cf21, could only be substituted by glutathione and glutathione disulfide with considerable loss in activity. The pronounced specificity of tryparedoxin is further accentuated by low limiting Km values for Cf21 and trypanothione (2.2 microM and 130 microM, respectively, as compared to 990 microM for gluthathione disulfide and an infinite value for glutathione). Tryparedoxin can therefore be classified as a trypanothione: peroxiredoxin oxidoreductase. The reduction of tryparedoxin by trypanothione appears to be the rate-limiting step in the trypanothione-dependent hydroperoxide reduction because(a) the regeneration of reduced tryparedoxin from the tryparedoxin-trypanothione complex is rate limiting (k[cat] 392 min[-1]), (b) the physiological trypanothione concentrations may not always saturate tryparedoxin, and (c) the rate constants for the net forward reaction of Cf21 are faster than those of the tryparedoxin reaction. The functional characteristics of tryparedoxin explain the limited capacity of trypanosomatids in coping with oxidative stress and qualify the enzyme as a potential target for the design of specific trypanocidal compounds.