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.
