Science and Technology Production
Congress
Authorship
LEROUX, ALEJANDRO EZEQUIEL
;
Maugeri, Dante
;
Cazzulo, Juan José
;
Nowicki, Cristina
Date
2008
Publishing House and Editing Place
Círculo Médico de Rosario
ISSN
0327-5019
Summary
Information provided by the agent in
SIGEVA
Two types of NADP-dependent isocitrate dehydrogenases have been characterized in higher eukaryotes: the mitochondrial and cytosolic (cNADP-IDHs) isozymes. Although the cNADP-IDH has a peroxisome targeting signal (PTS1) at its C-terminus, both a cytosolic and peroxisomal localization has been observed in eukaryotes. NADP-IDHs catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate with the concomitant production of NADPH, the essential coenzyme for biosynthetic and detoxification ...
Two types of NADP-dependent isocitrate dehydrogenases have been characterized in higher eukaryotes: the mitochondrial and cytosolic (cNADP-IDHs) isozymes. Although the cNADP-IDH has a peroxisome targeting signal (PTS1) at its C-terminus, both a cytosolic and peroxisomal localization has been observed in eukaryotes. NADP-IDHs catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate with the concomitant production of NADPH, the essential coenzyme for biosynthetic and detoxification reactions. In most organisms, the cNADP-IDH jointly with the two pentose-phosphate pathway dehydrogenases and the malic enzymes constitute the main cellular source of NADPH. Based on the TriTryps genomes, cNADP-IDHs are likely to be functional in trypanosomes, but not in Leishmania. Sequence alignment showed that T. cruzi and T. brucei putative cNADP-IDH are closely related (67 % identity) with the mammalian cNADP-IDHs. We cloned and functionally characterized a cNADP-IDH from T. cruzi. The N-terminal His-tagged enzyme was expressed in E. coli. The kinetic parameters showed that at least in vitro T. cruzi cNADP-IDH is notably more active in the direction of oxidative decarboxylation of isocitrate. Km values of 54 uM for isocitrate, 56 uM for NADP and 3.4 mM for 2-oxoglutarate were determined. Although the enzyme exhibits a PTS1 targeting signal, the cytosolic localization was confirmed by digitonine extraction experiments in T. cruzi and by immunofluorescence microscopy in T. brucei. Western-blot analysis showed that T. cruzi cNADP-IDH is expressed along the whole life cycle of this pathogen. Although the biological role of the cytosolic IDH is still uncertain, its contribution to NADPH production in the cytosol should not be dismissed.
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Key Words
isocitrato deshidrogenasatrypanosoma cruzi