Two metallocarboxypeptidases from the protozoan Trypanosoma cruzi belong to the M32 family, found so far only in prokaryotes

Abstract

MCPs (metallocarboxypeptidases) of the M32 family of peptidases have been identified in a number of prokaryotic organisms, and only a few of them have been characterized biochemically. Members of this family are absent from eukaryotic genomes, with the remarkable exception of those of trypanosomatids. The genome of the CL Brener clone of Trypanosoma cruzi, the causative agent of Chagas' disease, encodes two such MCPs, with 64% identity between them: TcMCP-1 and TcMCP-2. Both genes, which are present in a single copy per haploid genome, were expressed in Escherichia coli as catalytically active polyHis-tagged recombinant enzymes. Despite their identity, the purified TcMCPs displayed marked biochemical differences. TcMCP-1 acted optimally at pH 6.2 on FA {N-(3-[2-furyl]acryloyl)}-Ala-Lys with a K(m) of 166 muM. Activity against benzyloxycarbonyl-Ala-Xaa substrates revealed a P1' preference for basic C-terminal residues. In contrast, TcMCP-2 preferred aromatic and aliphatic residues at this position. The K(m) value for FA-Phe-Phe at pH 7.6 was 24 muM. Therefore the specificities of both MCPs are complementary. Western blot analysis revealed a different pattern of expression for both enzymes: whereas TcMCP-1 is present in all life cycle stages of T. cruzi, TcMCP-2 is mainly expressed in the stages that occur in the invertebrate host. Indirect immunofluorescence experiments suggest that both proteins are localized in the parasite cytosol. Members of this family have been identified in other trypanosomatids, which so far are the only group of eukaryotes encoding M32 MCPs. This fact makes these enzymes an attractive potential target for drug development against these organisms.

Unusual phyletic distribution of peptidases as a tool for identifying potential drug targets

“…In this issue of Biochemical Journal, Niemirowicz et al [1] report the discovery of novel peptidases from the protozoan Trypanosoma cruzi, the causative agent of Chagas' disease in Latin America. Niemirowicz et al [1] have been able to show that two genes are present in T. cruzi, encoding metal-dependent carboxypeptidases with different specificities and distributions during the protozoan life cycle.…”

“…Niemirowicz et al [1] have been able to show that two genes are present in T. cruzi, encoding metal-dependent carboxypeptidases with different specificities and distributions during the protozoan life cycle. Homologues have also been found in the closely related protozoan, Leishmania major.…”

“…An enzyme that is present in a parasite which is not only absent in its hosts, but its hosts have no homologues, makes the enzyme an ideal drug target, and Niemirowicz et al [1] suggest that the carboxypeptidases might be potential drug targets for the prevention of Chagas' disease.…”

“…Apart from the zooflagellate protozoa, the family is restricted to bacteria and archaea. The surprisingly high similarity between the Trypanosoma and Leishmania carboxypeptidases and those from bacteria has led Niemirowicz et al [1] to suggest that an ancient horizontal transfer of a gene took place, either after the separation of zooflagellates from other protozoa or the gene was lost in the ancestors of other protozoa.…”

“…Although Niemirowicz et al [1] may have identified an alternative point of attack, characterization of the carboxypeptidases is a long way from establishing a promising drug target. First of all, it is not known what the physiological functions of the carboxypeptidases are, and whether the protozoan can survive without these gene products.…”