Biochemical characterization of dihydroxyacetone kinase from Trypanosoma cruzi (TcDAK)

JUAN MATIAS VIECENZ; GARAVAGLIA, PATRICIA ANDREA; TASSO, LAURA MÓNICA; MAIDANA CRISTINA; GABRIELA ANDREA GARCIA; CANNATA, JOAQUÍN J B

Mar del Plata

Congreso; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; 2019

Sociedad Argentina de Investigación Clínica- Sociedad Argentina de Protozoología

Dihydroxyacetone (DHA) is used as carbon source by many cell types; however, at high concentrations, it is toxic and some microorganisms are susceptible to this compound. DHA detoxification is dependent on a functional dihydroxyacetone kinase (DAK) which converts DHA to DHA-phosphate (DHAP). Trypanosomacruzi, the etiological agent of Chagas disease, possess two genes encoding for putative ATP-dependent DAKs (TcDAKs). Previous studies have shown that T. cruziepimastigotes are able to grow in the presence of DHA and DAK activity was found in their lysates. Nevertheless, TcDAK has not been characterized so far. With this aim, recombinant TcDAK, expressed in E. coli by pJexpress vector and purified by IMAC, was used to determine kinetic properties of this enzyme. DAK activity was measured using DHA and ATP as substrates in the presence of 2 mM MgCl2 in a coupled assay by enzymatic reduction of DHAP with NADH in the presence of glycerol-3-P dehydrogenase. TcDAK exhibited Michaelis-Menten kinetics for DHA (Km=6.3 M and Vmax=12.79 moles/min/mg of protein). On the other hand, it showed sigmoideal kinetics for Mg-ATP (S0.5=125.03 M, Vmax=1.18 moles/min/mg of protein and Hill coefficient=1.99). TcDAK activity in the presence of other metals different than Mg++ was also evaluated. All of them gave lower activities than that observed with Mg-ATP (Ca++>Cd++>Mn++>Co++> Zn++). Additionally, TcDAK expression was evaluated in epimastigote and trypomastigote lysates by western blot using an anti-recombinant TcDAK serum. Epimastigotes and trypomastigotes showed the presence of two proteins recognized by the specific antiserumof molecular masses of 60 and 49 kDa, respectively, suggesting that trypomastigotes might have aTcDAK cleaved isoform. These results endorses the hypothesis that T. cruzi possess an active TcDAK which enables the parasite to use DHA as carbon source.