Metallocarboxypeptidase-1 and ribose 5-phosphate isomerase: two enzymes from Trypanosoma cruzi without orthologs in higher eukaryotes.

NIEMIROWICZ, GABRIELA TERESA; STERN, ANA LAURA; CAZZULO, JUAN JOSÉ

Armaçao dos Buzios, RJ

Congreso; XIII International Congress of Protistology; 2009

  Trypanosoma cruzi, the protozoan parasite which is the causative agent of the American Trypanosomiasis, Chagas Disease, contains at least three carboxypeptidases: a serine carboxypeptidase (TcSCP) belonging to the S10 family, and two metallocarboxypeptidases (TcMCPs) belonging to the M32 family, found up to now only in prokaryotes.  The two TcMCPs encoded by the genome of the CL Brener clone have 64 % identity between them.  Both genes are present as single copies per haploid genome, and are differentially expressed in the parasite’s life cycle.  Whereas TcMCP1 is expressed by the four main stages, TcMCP2 seems to be present only in the stages present in the insect vector.  The purified recombinant proteins differed also in specificity;  whereas TcMCP1 preferred a C-terminal basic residue, TcMCP2 preferred aromatic and aliphatic non polar residues.  TcMCP1 was able to degrade several important hormonal peptides, like bradikinin.   Immunofluorescence experiments showed that both TcMCPs are localized in the cytosol.  Both enzymes are dimers consisting of identical subunits.  The 3-D structure of the 128 kDa TcMCP1 was determined by X-Ray crystallography to a resolution of 2.0 Å.  The monomer resembles an elongated cowry shell, with a long, deep, narrow active-site cleft mimicking the aperture.   A residue identified as crucial to shape the S1’ pocket in TcMCP1, Met 304, was mutated to the respective residue in TcMCP2, an Arg, leading to a TcMCP1 variant with TcMCP2 specificity.  If the activity of TcMCP1 proves to be essential for the parasite, it would be a good candidate as a possible target for the development of new drugs against Chagas Disease, since the M32 family is absent from all eukaryotic genomes sequenced so far, with the only exception of trypanosomatids. Trypanosoma cruzi  contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B ribose 5-phosphate isomerase (Rpi), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).  4-Phospho-D-erythronohydroxamic acid (4PEH), an analogue to the reaction intermediate since the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. We have recently obtained the 3-D structure of the enzyme, in complexes with R5P or  4PEH, and of the C69A mutant with Pi at the active site, with resolution between 1.4 and 1.9 Å.  These results contribute to the knowledge of the mechanism of the Rpi reaction, and, since RpiB genes are absent from the genomes from higher eukaryotes, the knowledge of the active site will be useful for the design of inhibitors which may become lead compounds for the development of novel drugs against Chagas disease.