“Functional characterization of stage-specific aminotransferases from trypanosomatids”

MARCIANO DANIELA,; MAUGERI DANTE,; CAZZULO JUAN JOSÉ,; NOWICKI , CRISTINA

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

Elsevier

Año: 2009

0166-6851

Abstract: As part of a study on aminotransferases, genes coding for putative enzymes from Trypanosoma brucei and Leishmania major (alanine aminotransferases: ALATs, Tb927.1.3950 and LmjF12.0630;kynurenine aminotransferase: KAT, Tb10.389.1810; and tyrosine aminotransferase: TAT,LmjF36.2360) were cloned and functionally expressed in E. coli. The putative T. brucei KAT, in factcoded for a glutamine aminotransferase (GlnAT), which exhibited a notably high affinity (in themolar range) towards glutamine and cysteine; in addition, like bacterial GlnATs and mammalianKATs, it was able to utilize different 2-oxoacids as amino acceptors. L. major TAT resembled T.cruzi TAT in substrate specificity, although the leishmanial enzyme did not exhibit ALAT activity. On the other hand, T. brucei ALAT, shortened by the first 65 amino acids assigned in the data bases,was functional and actively transaminated the substrate pair L-alanine and 2-oxoglutarate.Moreover in Western blots, the molecular size of the protein detected in crude extracts of T. brucei procyclics was identical to the value of the recombinant enzyme. Like T. brucei and T. cruziorthologues, L. major ALAT displayed narrow substrate specificity. The leishmanial ALAT, like theT. cruzi enzyme, exhibited a dual subcellular localization, in the cytosol and in the mitochondrion. In line with the findings of comparative proteomic analyses of insect and mammalian stages of T.brucei and Leishmania parasites, our results also showed that T. cruzi ALAT is constitutivelyexpressed, with remarkably higher levels being detected in amastigotes than in epimastigotes.ALATs are expressed in the clinically important stages of TriTryps, probably fulfilling an essential role, which deserves further studies.