IMP specific 5' -nucleotidase 1: in vitro characterization and attempts at conditional knockdown in plasmodium falciparum

Abstract

Plasmodium falciparum is one of the five species of Plasmodium that infect humans to cause malaria, which is one of the leading causes of death in low-income countries. It possesses a highlyspecialized metabolism which enables it to occupy the anucleated erythrocytes of the host and derives energy mainly through the upregulation of glycolysis. Although it has the ability to synthesize pyrimidines de novo, the de novo purine synthesis pathway is absent in the parasite and therefore it obtains purines from the host via the purine salvage pathway. Purines are mainly salvaged in the form of hypoxanthine which is phosphoribosylated to form inosine monophosphate (IMP). These IMP pools acts as precursors for the nucleotides adenosine monophosphate and guanosine monophosphate. By catalyzing the hydrolysis of 5'-nucleotide mono-phosphates like IMP to nucleosides and inorganic phosphate, 5’-nucleotidases play a significant role in nucleotide metabolism. Even though the commonly known cN-II class of 5'-nucleotidases is absent in Plasmodium, recently, a homolog of Saccharomyces cerevisiae IMP specific 5'-nucleotidases (ISN1), belonging to the Haloacid Dehalogenase (HAD) family has been identified in Plasmodium falciparum. The first chapter of this thesis deals with an introduction to Plasmodium, its metabolism and the HAD superfamily of proteins to which ISN1 belongs. The second chapter describes the structure-function analysis of the protein. Earlier studies done in the lab with the recombinant form of this enzyme have shown that the enzyme preferentially hydrolyses IMP and is modulated by ATP. The crystal structure of this enzyme, the first from its class, has been solved recently through a collaborative effort. Based on the structure, the role of several conserved and non-conserved residues has been identified. These residues have further been mutated to validate their role in catalysis. Chapter two specifically discusses the role of one particular residue, F403, in catalysis and also reports the development of an ∆N-30 deletion construct of ISN1 that will aid in further crystallographic studies. Chapter three focuses on the in vivo characterization of ISN1. The studies described in this chapter are towards addressing the essentiality of the protein as well as the localization of the protein in the parasite. Chapter four summarizes our current understanding of the enzyme.