Metabolic significance of fumarate in plasmodium falciparum and biochemical and structural characterization of class I fumarate hydratase from plasmodium falciparum and methanocaldococcus jannaschii

Abstract

Energy metabolism in Plasmodium falciparum asexual stages is unique with respect to the set of pathways used to achieve the objective function of biomass production. ATP demand of the parasite is almost completely met by substrate level phosphorylation reactions of an unregulated glycolysis in the parasite with very low contribution from the tricarboxylic acid (TCA) cycle (Lang-Unnasch & Murphy, 1998; White et al., 1983). However, the TCA cycle in the intraerythrocytic stages of Plasmodium is completely operational with low flux and is largely anaplerotic in nature (Cobbold et al., 2013; MacRae et al., 2013). Anaplerotic contribution to the TCA cycle is mainly from three metabolites viz., phosphoenolpyruvate (PEP) derived from glycolysis, glutamine (derived from hemoglobin degradation and from host), and fumarate derived from purine nucleotide cycle (PNC). Purine nucleotide cycle is a set of reactions in the parasite cytosol, catalysed by three enzymes adenylosuccinate synthetase (ADSS), adenylosuccinate lyase (ASL) and adenosine monophosphate deaminase (AMPD), that involve the cyclic interconversion of IMP to AMP and the simultaneous conversion of one molecule of aspartate to one molecule of fumarate and ammonia. The fumarate generated enters the mitochondria and gets converted to malate and then to oxaloacetate through sequential action of the enzymes fumarate hydratase (FH) and malate-quinone oxidoreductase (MQO), the classical reactions of TCA cycle (Bulusu, Jayaraman, & Balaram, 2011). Oxaloacetate generated can have multiple fates in the cytosol. Through a reverse genetic approach, the anaplerosis through PEP has been shown to be important for the asexual growth of the parasite (Storm et al., 2014). In a separate study it was shown that all the genes of the TCA cycle can be knocked out except for two, FH and MQO, implying two facts; (i) the essentiality of the enzymes FH and MQO and (ii) the dispensability of glutamine mediated anaplerosis(Ke et al., 2015). The metabolic significance of fumarate anaplerosis is still not clear. In this work, we intended to study the metabolic significance of fumarate generated from PNC and biochemical characterisation of the first enzyme in fumarate metabolism, fumarate hydratase. The thesis is broadly divided into two sections: the first section of the thesis attempts to address the metabolic significance of fumarate generated from PNC. This section also deals with the development of a methodology to trace the fate of ammonia which subsequently can be used to trace the α-amino group of aspartate that feeds into the PNC. The second section deals with the biochemical and structural characterisation of fumarate hydratase from P. falciparum and Methanocaldococcus jannaschii.