Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi

GARAVAGLIA, PA; RUIZ, AM; DURAN, R; IBARRA, SE; GARCIA, GA

Mendoza, Prov. Mendoza, Argentina

Congreso; VII Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2005

Sociedad Argentina de Protozoologia

Drugs currently used for treatment of Trypanosoma cruzi infection, the ethiological agent of Chagas’ disease, have shown inadequate side effects and variable efficiency. With the aim to describe new putative targets for trypanocidal drug design we attempted to identify novel reductases in the parasite. As the dye Cibacron Blue F3 is capable of binding strongly many enzymes requiring adenylyl-containing  substances, proteins from the epimastigote stage of T. cruzi bound to Cibacron Blue-Sepharose were specifically eluted with NADPH. A main protein with an apparent molecular weight of 32 kDa was obtained which co-eluted with other four minor proteins. MALDI-TOF analysis of this protein determined it belongs to the aldo-keto reductase (AKR) superfamily, so it was denominated TcAKR. Amino acid sequence analysis shows a 59% and 58 % of identity with prostaglandin F synthase  from Trypanosoma brucei and 2.5 Diketo-D-gluconic acid reductase from Bacillus cereus, respectively. TcAKR gene was amplified by PCR and cloned into pQE30 vector. The recombinant TcAKR purified by IMAC showed NADPH dependent reductase activity with 4-nitrobenzaldehyde, the most commonly used AKR substrate. AKR superfamily involves related enzymes that share structural features, but because of their overlapping substrate specificities, they may participate in diverse metabolisms. TcAKR is the first AKR described in T. cruzi, so its biochemical characterization will allow to elucidate its physiological function in the parasite. In addition, as TcAKR has no homology to mammalian enzymes, it will be worth determining if this protein can be postulated as a target enzyme for trypanocidal compounds.