30 and TcME2b, Gene ID Tc00.1047053508647.280), T. brucei congolense putative MEs (TcongME1, Gene ID TcIL3000.11.5690 and TcongME2, Gene this website ID TcIL3000.11.5680), T. brucei gambiense putative MEs (TbgME1, Gene ID Tbg972.11.6150 and TbgME2, Gene ID Tbg972.11.6140) and T. vivax putative MEs (TvivME1, Gene ID TvY486_1105630 and TvivME2, Gene ID TvY486_1105620). Sequence numbering corresponds to MAOM_HUMAN. Strictly conserved residues in all the compared sequences

are indicated in red whereas those partially conserved or equivalent are indicated in blue. Those residues which were identified in the crystallographic structures of the mammalian and pigeon MEs to be involved in divalent cation, malate and cofactor NAD(P) binding (Chang & Tong, 2003) are highlighted in black, green and grey backgrounds, respectively. In addition, the residues involved in the catalytic mechanism are highlighted in blue backgrounds with white lettering. The strictly conserved Lys residue which determines the coenzyme specificity in the NADPH dependent enzymes is indicated by a triangle (▴); this residue is substituted for Gln in the

NAD linked-enzymes. The strictly conserved phenylalanine residue from the N-terminal region recognized in higher eukaryotes to participate in the subunit interaction is indicated by a circle (•). The NADB_Rossmann typical sequence motif is indicated as GXGXXG and GXGXXAXXXA. Fig. S2. Heterologous expression and purification of ME isozymes from Trypanosoma cruzi and Trypanosoma brucei in Escherichia coli cultures. The recombinant Histagged enzymes were expressed and purified as described in Section PD-0332991 nmr 2. Each of the recombinant proteins (5 μg) were subjected to SDS-PAGE

in 7.5 % polyacrylamide gels, under reducing conditions and were visualized by Coomassie Blue staining. Lane1, T. brucei TbME1; Lane 2, TbME2; Lane 3, TcME2; Lane 5, TcME1; Lane 6, molecular weight markers, the corresponding values in kDa are shown on the right side of the panel. Fig. S3. Immunological cross-reactivity of the recombinant MEs from Trypanosoma brucei. Equal Olopatadine amounts (100ng) of the recombinant ME1 (lanes 1 and 3) and ME2 (lanes 2 and 4) from T. brucei were resolved by SDS-PAGE on 7.5% polyacrylamide gels and transferred by electro-blotting to nitrocellulose membranes (Panels A and B). The recombinant proteins were also applied in native conditions on nitrocelulose membranes (Panels C and D); 10 and 100 ng of each isozyme were dotted as depicted in the figure. The blotted samples were developed with specific polyclonal antiserum raised against each of the recombinant isozymes (Panels A and C, anti-TbME1 serum; Panels B and D anti-TbME2 serum). Fig. S4. Immunological cross-reactivity of the recombinant MEs from Trypanosoma cruzi. Equal amounts (100ng) of the recombinant ME1 (lanes 1 and 3) and ME2 (lanes 2 and 4) from T. cruzi were resolved by SDS-PAGE on 7.5% polyacrylamide gels and transferred by electro-blotting to nitrocellulose membranes (Panels A and B).