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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 Chamond^§, Christophe Grégoire^§¶, Nicolas Coatnoan, Catherine Rougeot, Lucio Holanda Freitas-Junior, José Franco da Silveira^**, Wim M. Degrave, and Paola Minoprio^§§

From the Departments of  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  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 K_m of the enzyme isoforms were of a similar order of magnitude (29-75 mM), V_max varied between 2 × 10⁴ and 5.3 × 10⁵ 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.

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

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