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J. Biol. Chem., Vol. 278, Issue 18, 15484-15494, May 2, 2003
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Biochemical Characterization of Proline Racemases from the Human Protozoan Parasite Trypanosoma cruzi and Definition of Putative Protein Signatures*

Nathalie ChamondDagger §, Christophe GrégoireDagger §, Nicolas CoatnoanDagger , Catherine RougeotDagger , Lucio Holanda Freitas-Junior||, José Franco da Silveira**, Wim M. DegraveDagger Dagger , and Paola MinoprioDagger §§

From the Departments of Dagger  Immunology and || Parasitology, Institut Pasteur, Paris, 75724, France, ** Department of Microbiology, Immunology and Parasitology, Unifesp/Escola Paulista de Medicina, Sao Paulo 04023-062, Brazil, and Dagger Dagger  Department of Biochemistry and Molecular Biology, Instituto Oswaldo Cruz, Rio de Janeiro 21045-900, Brazil

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 × 10-4 and 5.3 × 10-5 mol of L-proline/s/0.125 µM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.


* This work was supported in part by Institut Pasteur and CNRS URA 1960.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY 1409447.

§ Contributed equally to this work.

Fellow of the Pasteur Institute.

§§ To whom correspondence should be addressed. Tel./Fax: 33-1-45-68-86-15; E-mail: pmm@pasteur.fr.


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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