and NADPH consumption was followed at 340 nm. All reactions were performed at 25uC and monitored with a Shimadzu UV- 2401 PC spectrophotometer (Shimadzu Corporation). Phylogenetic analysis of TXN sequences TXN sequences were obtained at the TriTrypDB (http:// tritrypdb.org/tritrypdb/), except for the C. fasciculata sequences, which were recovered from the NCBI webpage (http://www.ncbi. nlm.nih.gov/nuccore). TXNs designation follows the suggested genetic nomenclature for Trypanosoma and Leishmania [48], mentioning the species abbreviation and the sequence number. Systematic names attributed by TriTrypDB to each gene are listed next (May 2010). LbTXN1a, LbrM29_V2.1240; LbTXN1b, LbrM29_V2.1230; LbTXN2, LbrM29_V2.1220; LbTXN3, LbrM31_ V2.2190; LmTXN1, LmjF29.1160; LmTXN2, LmjF29.1150; LmTXN3, LmjF31.1960; LmTXN4, LmjF31.1970; LmxTXN1, LmxM08_29.1160; LmxTXN2, LmxM08_29.1150; LmxTXN3, LmxM30.1960; LmxTXN4, LmxM30.1970; LiTXN1, LinJ29_ V3.1250; LiTXN2, LinJ29_V3.1240; LiTXN3, LinJ31_ V3.2000; LiTXN4, LinJ31_V3.2010; TbTXN1a, Tb927.3.3780; TbTXN1b, Tb927.3.3760; TbTXN2, Tb927.3.5090; TgTXN1a, Tbg972.3.4110; TgTXN1b, Tbg972.3.4090; TgTXN2, Tbg972.3. 5720; TcTXN1, Tc00.1047053509997.30; TcTXN2, Tc00. 1047053509997.20; TcoTXN1, TcIL3000.0.52740; TcoTXN2, TcIL3000.0.52730; TvTXN1, TvY486_0303158; TvTXN2, TvY486_0303156 (note that some are temporary names). In the case of C. fasciculata sequences the accession numbers are as follows: CfTXN1, AF084456; CfTXN2, AF055986). Nucleotide sequences were translated, aligned at the amino acid level with ClustalW (www.ebi.ac.uk/Tools/clustalw/) and the resulting alignments used as a guide to obtain the final nucleotide alignment. Phylogenetic trees were constructed by the MEGA4 program [29]. Statistical analysis Statistical significance was analyzed by independent-samples t- test, using SPSS (Version 16.0). Supporting Information Text S1 Sequence analysis of TXN-like proteins predicted in the L. infantum genome that do not specify active tryparedoxins. Found at: doi:10.1371/journal.pone.0012607.s001 (0.17 MB PDF) Text S2 Exchange of Arg40 by a Ser does not allow LiDTXN3 to recover TXN activity. Found at: doi:10.1371/journal.pone.0012607.s002 (0.13 MB PDF) Figure S1 Sequence analysis of LiTXN4, LiTXN5, LiTXN6 and LiTXN7. A. Nucleotide and deduced amino acid sequence of LiTXN4. Residues in the grey background represent the Trp-Cys- Pro-Pro-Cys-Arg active site signature of TXNs. The start and stop codons are underlined, showing that LiTXN4 is a pseudogene. B- D. Sequence alignment of LiTXN5 (B), LiTXN6 (C) and LiTXN7 (D) with LiTXN1 and LiTXN2 (the latter two considered here as a distinct subgroup). Strict identity across all sequences is represent- References Mitochondrial Tryparedoxins ed with white letters on a black background. Similarity across subgroups is marked with black frames. Residue similarity in each subgroup is indicated with bold letters. Differences between subgroups are marked with black frames and grey background. The TXN active site motif is highlighted with a bar. Residues involved in the hydrogen bond system are marked with stars and those implicated in reaction with trypanothione with arrowheads. The acidic area, important for interaction with 2-Cys PRXs, is marked with diamonds. The C-terminal hydrophobic tail of LiTXN5, predicted to specify a transmembrane domain (by bioinformatics analysis with TMpred and TopPred), is marked on light grey background. Sequence numbering refers to LiTXN1. The secondary structural elements (b1–7 = beta strands; a1– 4 = alpha helices; g1–3 = 310 helices; TT = turns) deduced for LiTXN1, according to the crystal structure of recombinant C. fasciculata TXN1 (PDB ID 1QK8) is shown in D. The predicted amino acidic sequences of the LiTXN7 counterparts in T. brucei (TbTXN3) and T. cruzi (TcTXN3) (Gene IDs: Tb927.10.3970 and Tc00.1047053506959.20, respectively, according to the Tri- TrypDB) are also aligned in D. Found at: doi:10.1371/journal.pone.0012607.s003 (0.41 MB PDF) Figure S2 Analysis of LiDTXN3R40S enzymatic activity. A. Formation of the insoluble insulin B chain (monitored at 650 nm) by DTT-reduced LiDTXN3 (filled squares) or LiDTXN3R40S (open squares), or by NADPH/TR/T(SH)2- reduced LiDTXN3 (filled circles) or LiDTXN3R40S (open circles). Negative controls were performed in the same reaction mixtures [either with DTT or with the TR/T(SH)2 redox system] without any addition of TXN (triangles). B. Reduction of LiDTXN3R40S by T(SH)2 followed by monitoring NADPH consumption at 340 nm. The reaction systems contained NADPH, TR and T(SH)2 as reductants for TXN and insulin as final electron acceptor. The TXNs tested were LiDTXN3 (filled squares) and LiDTXN3R40S (open squares). Negative control contains no TXN (triangles). C. Reduction of TXN-dependent peroxidases by LiDTXN3R40S. Reaction mixtures consisted in NADPH, TR, T(SH)2, recombi- nant TXN [either LiDTXN3 (closed symbols) or LiDTXN3R40S (open symbols)] and four different peroxidases, namely LimTXNPx (triangles), LicTXNPx1 (squares), LicTXNPx2 (dia- monds) and LinsGPXA1 (circles). Reactions were initiated by addition of H2O2 and NADPH consumption was followed at 340 nm. Found at: doi:10.1371/journal.pone.0012607.s004 (0.10 MB PDF) Acknowledgments We acknowledge Jorge Vieira and Cristina Vieira for helpful discussions on evolutionary analysis and Simon Lee for revising the English in the manuscript. We thank Mary E. Wilson and Andre´ Schneider for the anti- gp63 and the anti-TbCytc antibodies, respectively. Author Contributions Conceived and designed the experiments: HC SR AMT. Performed the experiments: HC SR SC FT CS. Analyzed the data: HC SR AMT. Wrote the paper: HC SR AMT. 1. Krauth-Siegel RL, Comini MA (2008) Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism. Biochim Biophys Acta 1780: 1236–1248. 2. Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe´ L (1997) A unique cascade of oxidoreductases catalyses trypanothione-mediated peroxide metab- olism in Crithidia fasciculata. Biol Chem 378: 827–836. 3. Wilkinson SR, Meyer DJ, Taylor MC, Bromley EV, Miles MA, et al. (2002) The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin. J Biol Chem 277: 17062–17071. 4. Hillebrand H, Schmidt A, Krauth-Siegel RL (2003) A second class of peroxidases linked to the trypanothione metabolism. J Biol Chem 278: 6809–6815. PLoS ONE | www.plosone.org 11 September 2010 | Volume 5 | Issue 9 | e12607