Identification and biochemical characterization of an ATP-dependent dihydroxyacetone kinase from Trypanosoma cruzi

VIECENZ, JUAN MATÍAS; GARAVAGLIA, PATRICIA ANDREA; TASSO, LAURA MÓNICA; MAIDANA, CRISTINA GRACIELA; BAUTISTA CANNATA, JOAQUÍN JUAN; GARCÍA, GABRIELA ANDREA

EXPERIMENTAL PARASITOLOGY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Año: 2021 vol. 231 p. 108178 - 108178

0014-4894

Abstract:Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the aethiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmoles/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 μM for DHA and a K0.5 of 124.7 μM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.