Identification, cloning and characterization of an aldo-keto reductase from Trypanosoma cruzi with quinone oxidoreductase activity

GARAVAGLIA, PA; CANNATA, JB; RUIZ, AM; MAUGERI, D; BUA, J; GALLEANO, M; DURAN, R; GARCIA, GA

Armacao dos Buzios, Río de Janeiro- Brasil

Congreso; XIII Congreso Internacional de Protistología (ICOP), XXXVI Encuentro Anual sobre Investigación Básica en Enfermedad de Chagas; 2009

Sociedad Brasilera de Protistologia

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 parasitic enzymes involved in the action mechanisms of trypanocidal drugs, we attempted to identify novel NADPH-dependent oxido-reductases from T. cruzi, since it has been reported that reductases are crucial in this metabolism. The passage of soluble fraction of epimastigote lysates through Cibacron Blue-Sepharose followed by NADPH elution yielded a predominant protein with an apparent molecular weight of 32 kDa identified by MALDI-TOF as an aldo-keto redutase superfamily member (TcAKR). The release profile of TcAKR from intact epimastigotes by titration with digitonin suggested that TcAKR is mainly localized in the cytosol. We confirmed by western blot that TcAKR is also present in trypomastigote/amastigote lysates. The TcAKR gene was cloned in E. coli and the recombinant protein (recTcAKR) showed NADPH-dependent reductase activity with most common AKR substrates, 4-NBA and 2-DHA, with a substrate saturation curve consistent with the Michaelis-Menten model. No activity was detected with NADH as cofactor. We also tested whether recTcAKR may reduce naphtoquinones (NQ), since many of these compounds have displayed important trypanocidal activity. recTcAKR showed reductase activity with o-NQ (1,2 NQ, 9,10 PQ and b-lapachone) with concomitant generation of free radicals, but did not exhibit affinity for p-NQ (5H-1,4-NQ, 2H-1,4-NQ, a-lapachone and menadione). The substrate saturation curve with o-NQ fitted to a sigmoidal curve, suggesting that recTcAKR presents a cooperative behavior. In agreement with this hypothesis, three peaks assigned to monomers, dimers and tetramers were obtained when recTcAKR was submitted to a Superose 12 gel chromatography column. Our results indicate that TcAKR, the first member of the AKR family described in T. cruzi, may participate in the action mechanisms of trypanocidal drugs.