Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

ABSTRACT

The intracellular parasite Trypanosomacruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite’s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzithroughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number 2.7.7.1). The identification of the NMNAT of T. cruziis important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruziusing bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.