, 2009). Based on these data, we evaluated how heme A is synthesized by T. cruzi (and the other trypanosomatids). The coding sequences for putative proteins homologous to HOS and HAS have been identified in the T. cruzi genome. One cds, Tc00.1047053511211.70, was identified as a HAS homologue (named TcCOX15 and TcCox15 for the corresponding protein). Two cds were associated with HOS (Tc00.1047053509601.59 and Tc00.1047053509767.59)

presenting a sequence identity of 98% (named TcCOX10A and TcCOX10B, and TcCox10 A and B for the corresponding protein sequences). The predicted protein sequences [TcCox10 (A and B) and TcCox15] show about 52% and 56% homology and 37% and 41% identity to their S. cerevisiae orthologues, and they are also conserved in other trypanosomatids Pexidartinib mouse (Fig. 1). The multiple sequence alignment of HOSs includes the available trypanosomatid putative protoheme IX farnesyltransferase (HOS) and the S. cerevisiae Cox10 protein (Fig. 1a). The residues N196, R212, R216 and H317 (S. cerevisiae numbering), which are involved in the protein’s function (Bestwick et al., 2010), are conserved in trypanosomatid sequences (indicated in Fig. 1a). The multiple sequence alignment of HAS proteins includes the available trypanosomatid putative HAS enzymes and the S. cerevisiae Cox15

protein (Fig. 1b). The alignment shows that residues involved in HAS activity based on studies from selleck chemical the Bacillus subtilis CtaA enzyme are also conserved in trypanosomatid sequences (Barros et al., 2001; Hederstedt et al., 2005). also Figure 1b shows the residues

H169, H245 and H393 from S. cerevisiae numbering, which correspond to CtaA H60, H123 and H216, respectively. Both T. cruzi putative proteins present eight predicted TMs, which is compatible with this type of protein (Fig. 1). The cds for TcCOX10 and TcCOX15 were amplified by PCR using total genomic DNA as the template and introducing a 3′-coding sequence for a 6xHis tag. As TcCOX10 A and B cds show 98% identity, the primers designed recognize both of them equally. The amplified product for TcCOX10 coincided with the Tc00.1047053509601.59 (TcCOX10A) sequence, and is named TcCOX10 and TcCox10 hereafter for the corresponding protein. Both cds (TcCOX10 and TcCOX15) were subcloned into yeast expression vectors and used to transform yeast cells lacking the corresponding genes (Δcox10 and Δcox15). These knockout cells present a respiration-deficient phenotype due to the absence of heme A production and consequently a functionally inactive CcO complex (Nobrega et al., 1990; Glerum et al., 1997). This deficiency impairs the growth in a nonfermentable carbon source such as glycerol–ethanol, but they all can grow in a media containing a fermentable carbon source as glucose. Their respiratory function was restored once TcCOX10A.6xHIS or TcCOX15.6xHIS was expressed in Δcox10 or Δcox15, respectively (Fig. 2a). Both mutants were also transformed with plasmids containing the corresponding S.