Mitochondrial Tryparedoxins Figure 4. Sequence alignment of LiTXN3 with other TXNs. The first three sequences in the alignment refer to the TXN3 enzyme of L. infantum (Li) and to the TXN2 molecules of both T. cruzi (Tc) and T. brucei (Tb), all of which possess C-terminal hydrophobic tails (marked on light grey background), predicted to be transmembrane domains (by bioinformatics analysis using TMpred and TopPred). The remaining four sequences are molecules characterized previously and known to display classical TXN activity. Strict identity across all sequences is represented with white letters on a black background. These include the TXNs active site signature (highlighted with a bar). Similarity across both subgroups is marked with black frames. Residue similarity in each subgroup is indicated with bold letters. Residues involved in the hydrogen bond system are marked with stars. Residues implicated in reaction with trypanothione are marked with arrowheads (Arg134 is also likely involved in binding to 2-Cys PRXs). The acidic area, important for interaction with 2-Cys PRXs, is marked with diamonds. Sequence numbering refers to LiTXN3. Gene IDs attributed by TriTrypDB (May 2010) are as follows: LiTXN3, LinJ31_V3.2000; TcTXN2, Tc00.1047053509997.20; TbTXN2, Tb927.3.5090; LiTXN1, LinJ29_V3.1250; LiTXN2, LinJ29_V3.1240; TcTXN1; Tc00.1047053509997.30; TbTXN1, Tb927.3.3780. doi:10.1371/journal.pone.0012607.g004 Figure 5. LiTXN3 displays oxidoreductase but not tryparedoxin activity. A. Formation of the insoluble insulin B chain (monitored at 650 nm) by DTT-reduced LiDTXN3 (squares) or LiTXN2 (diamonds), or by LiDTXN3 reduced in the presence of NADPH, TR and T(SH)2 (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 LiDTXN3 by T(SH)2 followed by monitoring NADPH consumption at 340 nm. Reaction systems contained NADPH, TR and T(SH)2 as TXN reductant and insulin as final electron acceptor. The TXNs tested were LiDTXN3 (squares) and LiTXN2 (diamonds). Negative control contains no TXN (triangles). C. Reduction of TXN-dependent peroxidases by LiDTXN3. Reaction mixtures consisted in NADPH, TR, T(SH)2, recombinant TXN [either LiDTXN3 (closed symbols) or LiTXN2 (open symbols)] and four different peroxidases, namely LimTXNPx (triangles), LicTXNPx1 (squares), LicTXNPx2 (diamonds) and LinsGPXA1 (circles). Reactions were initiated by addition of H2O2 and NADPH consumption was followed at 340 nm. doi:10.1371/journal.pone.0012607.g005 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12607