Molecular Parasitology and
Protein Engineering



The primary interest of the lab is in studying essential metabolic pathways in the malaria parasite Plasmodium falciparum, with the aim to characterize them as drug targets against malaria.
Research has focused on enzymes of three pathways in the parasite:

Purine salvage pathway

Enzymes under study are:

1.Hypoxanthine Guanine phosphoribosyltranseferase (HGPRT)

HGPRT catalyses the transfer of a phosphoribosyl moiety from phosphoribosyl pyrophosphate to N9 of the purine base (Hypoxanthine, guanine or xanthine) to give the corresponding nucleotide,IMP, GMP or XMP. Despite high sequence and structural similarity, the human and parasite enzymes differ in their substrate specificities. The human enzyme can phosphoribosylate hypoxanthine and guanine while the P.falciparum enzyme has an additional activity on xanthine. This difference in substrate specificity indicates that there exist subtle variations in the active site geometry of the two enzymes, differences that are not very apparent in the crystal structures.
Three approaches have been used to address the question of substrate specificity in HGPRTs:

1)Study of the wild type human and Plasmodium falciparum enzymes
2)Characterization chimeric HGPRTs consisting of sequences from P.falciparum and human proteins
3)Generation of altered specificity variants by random and site-directed mutagenesis.
The extended substrate specificity of P. falciparum HGPRT could be exploited to develop parasite specific drugs that act through this enzyme. Molecules that are either inhibitors of HGPRT or function as purine analogs undergoing phosphoribosylation could be potential antiplasmodial agents. Toxicity of the phosphoribosylated purine analogs arises, either from their inhibition of down stream enzymes in the purine salvage pathway or from their eventual incorporation in to nucleotide pools. A screening methodology using functional complementation in E.coli, has been developed in this laboratory, to identify molecules that function as either HGPRT inhibitors or substrate analogs that undergo phosphoribosylation.

2. Adenylosuccinate synthetase (AdSS)

AdSS, the enzyme that follows HGPRT in the purine salvage pathway to AMP, catalyses the condensation of IMP with aspartate to give adenylosuccinate which is cleaved by adenylosuccinate lyase to AMP. The reaction is accompanied by the hydrolysis of GTP to GDP and requires Mg2+. AdSS and therefore the adenylosuccinate pathway to AMP is absent in the human erythrocyte making this enzyme a potential therapeutic target. The crystal structure of AdSS has been solved to 2Å, in collaboration with Prof.M.R.N Murthy, MBU, IISc. Features of the parasite enzyme identified from the crystal structure include the occurrence of a large number of positively charged residues and at least one ionized/oxidised cysteines at the dimer interface.


The glycolytic enzyme under study is P.falciparum triosephosphate isomerase (PfTIM), on which extensive structural and biochemical analyses have been carried out in collaboration with Prof.M.R.N.Murthy and Prof.P.Balaram of Molecular Biophysics Unit, Indian Institute of Science. The intra-erythrocytic stages of P.falciparum lack a functional tricarboxylic acid cycle and glycolysis serves as a major source of ATP for this organism. Hence, enzymes from the glycolytic pathway are attractive targets for antimalarial chemotherapy.
Triosephosphate isomerase exits as a dimer and PfTIM has been used as a model system for studying stability and unfolding of dimeric (a/b)8 barrel structures. PfTIM has been shown to have a robust structure and even in 8M urea, the dimers do not dissociate completely. Considerable levels of secondary and tertiary structure are still retained under such conditions. The importance of the residues in the dimer interface in conferring stability to both quaternary and tertiary structure of PfTIM has been extensively investigated by site-directed mutagenesis. These studies show that tinkering with residues at the interface drastically alters the stability of the dimer.
Micro-environments in PfTIM have also been examined, using single tryptophan mutants of this enzyme, by both steady state and time resolved fluorescence spectroscopy. These studies indicate that the environment around the tryptophan in the catalytic loop6 is less dynamic than that around the tryptophan in the dimer interface.

Proteolytic enzymes in P. falciparum:

Proteases play important roles in various events during the asexual, intraerythrocytic phase of the life cycle of P. falciparum. We have developed a novel multi-specificity peptide substrate to monitor different protease activities in lysates of P. falciparum. Protease activities on the peptide substrate are monitored by fluorescence resonance energy transfer (FRET) and efficient quenching of fluorescent donor and acceptor groups has been achieved by introduction of a beta-turn..
Studies using the FRET peptide substrate has been extended to examine the susceptibility of beta-amino acid containing peptides to proteolysis by plasmepsin II, an aspartic protease from P. falciparum.

   Last modified date: 01-06-2010