Cloning and biochemical characterization of three different malate dehydrogenase (mdh) isozymes from Leishmania major.

FLEMING-CÁNEPA, X.; LEROUX, ALEJANDRO E.; ARANDA, A.; CAZZULO, JUAN JOSE; NOWICKI CRISTINA

Puerto Iguazú

Congreso; XL Reunión Científica anual SAIB; 2004

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Recently we have shown that the expression of the MDH isozymes is developmentally regulated during the life cycle of Trypanosoma brucei. The cytosolic MDH is the only isozyme expressed in the bloodstream forms, and it seems to be even more abundant than in the procyclics. Like T. brucei, and unlike Trypanosoma cruzi, Leishmania mexicana also appears to possess at least three different MDHs. Due to the low abundance and stability of the natural enzymes we decided to investigate the functionality of the three ORFs identified in the L. major genome project. The nucleotide sequence encoding each of the putative MDHs was amplified by PCR and cloned into an expression vector, pET28. Sequence alignments and the presence of a PTS in one isoform suggest that one of the MDHs may be located in the mitochondrion, another in the glycosome and the third would be cytosolic. The functionality of the three MDHs was proved by heterologous expression in Escherichia coli. The potential gMDH had app. Km values for oxaloacetate and NADH of  0.03 mM and 0.05 mM, respectively. These kinetic parameters are almost identical for the other isozymes. However, the app. Vmax values obtained for cMDH and mMDH are nearly 20 fold higher than those obtained for the gMDH suggesting that the latter isoform is the least efficient for oxaloacetate reduction. Additionally, these results clearly distinguish T. brucei and L. major  from T. cruzi, since in the latter parasite only  mMDH and gMDH are present.