CLONING AND BIOCHEMICAL CHARACTERIZATION OF BROAD SPECIFICITY AMINOTRANSFERASES FROM TRYPANOSOMES

MARCIANO DANIELA; LLORENTE CONSTANZA; DE LA FUENTE CANDELARIA; CAZZULO JUAN JOSÉ; NOWICKI CRISTINA

Rosario, Santa Fe, Argentina

Congreso; XLII Reunion Anual Sociedad de Investigación en Bioquímica y Biología Molecular; 2006

Sociedad de Investigación en Bioquímica y Biología Molecular

  The gluconeogenic pathway has been recently demonstrated to be essential for virulence of Leishmania sp., aspartate being utilized among other amino acids for de novo syntheses of carbohydrates. These findings fit in well with our recent observation that a cytosolic aspartate aminotransferase with broad substrate specificity is expressed in all the biological cycle stages of L. mexicana. In the present work, we report the cloning and functional expression in E. coli cultures of cytosolic and mitochondrial aspartate aminotransferases (ASATs) from T. cruzi and T. brucei. The nucleotide sequences encoding the putative enzymes, identified by Blast searching in the genome data-bases, were amplified by PCR and cloned into pET28 vector. Upon purification to electrophoretic homogeneity, the recombinant T. cruzi and T. brucei putative cytosolic-type ASATs were able to transaminate actively aspartate in addition to aromatic amino acids and methionine. Leucine was also utilized, although less efficiently. Conversely, the putative mitochondrial-type ASATs specifically recognized the substrate pair aspartate/2-oxoglutarate. As judged from Western blot analyses, the cytosolic-type ASATs are expressed in all the stages of T. cruzi and T. brucei. These broad specificity enzymes might be essential along the whole life cycle of trypanosomatids, very likely playing a conserved function.