title: 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.
authors.affiliation: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Universidad Nacional de General San Martín-CONICET, Av. General Paz 5445, 1650 San Martín, Buenos Aires, Argentina.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Niemirowicz
authors.forename: Fabiola
authors.initials: F
authors.lastname: Parussini
authors.forename: Fernán
authors.initials: F
authors.lastname: Agüero
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Cazzulo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000201:enzymology; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; D002268:Carboxypeptidases