The two Malic isozymes of Trypanosoma brucei are expressed along the whole life cycle of parasite.

LEROUX, ALEJANDRO E.; NOWICKI, CRISTINA

Rosario

Congreso; XLII Reunión Científica anual SAIB; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Trypanosoma brucei is the causative agent of some of the most neglected diseases such as sleeping sickness. We have previously reported that the cytosolic malate deshydrogenase (cMDH) is the unique MDH expressed in the mammalian stages of this parasite. It has been postulated that cMDH might provide the substrate required by malic enzyme (ME) to synthesize NAPDH, an essential coenzyme for biosynthetic processes and oxidative stress defense. Two different sequences coding for putative malic enzymes have been identified by Blast searching of the T. brucei genome data bases, the putative ORFs were recognized as Tb11.02.3130 (EMTb1) and Tb11.02.3120 (EMTb2). The nucleotide sequence encoding each of the EMs were amplified by PCR and cloned into an expression vector, pET28. The functionality of the two MEs was proved by heterologous expression in Escherichia coli. Both MEs showed similar apparent Km values, when malate and NADP were assayed, Mn2+ being the preferred metal ion. However, the catalytic efficiency of EMTb1 is nearly 10 fold higher than that of EMTb2. EMTb1 and EMTb2 were evidenced by western-blotting in the crude extracts of procyclics and bloodstream forms. Therefore, our results demonstrate that T. brucei express two functional MEs in the adaptive forms of the insect and mammalian stages.