Molecular and Biochemical Characterization of Transketolase of Plasmodium falciparum

Abstract

Malaria caused by Plasmodium falciparum (P. falciparum), a brutal killer, takes lives of about two million people every year and unfortunately most of them are non-immune children under the age of five from sub-Saharan Africa. World’s 40% population is living under the threat of malaria and this disease holds third position after Pneumonia and Tuberculosis, in terms of mortalities among the infectious diseases. An estimated 300-500 million cases of malaria each year result in imposition of a huge economic burden on both, families and governments through lost productivity, missed education and high health care costs. In the absence of effective vaccines, chemotherapy and chemoprophylaxis remain the main operationally, administratively and financially feasible method of malaria control. Moreover, this limited armory is now greatly compromised by the spread of drug resistant parasite strains across the malaria endemic map. The increasing resurgence of P. falciparum malaria linked to the resistance, both of mosquitoes and parasites to various insecticides and conventional drugs, respectively calls for new therapeutic approaches to control this endemic disease. In continuation to malaria management, an international programme was launched in 1996 to sequence P. falciparum genome with the expectation that the genome sequence would open new avenues for malaria control. Genome sequencing of the malaria parasite has been completed. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A+T)-rich genome sequenced to date. The complete genome sequence has rewritten the entire metabolic directory of the malaria parasite and vividly exposed the metabolic differences between the host and parasite. Moreover, completed genome-sequencing project paved the path for post-genomic era, which revolutionized the malaria research with the advances in the technologies such as DNA Microarray and proteomics. Analysis using these techniques provides an excellent opportunity to select essential targets for new interventions by studying the complete spectrum of events taking place at a time during the biologically important processes such as host cell invasion or drug resistance. Interfering with parasite specific metabolic pathways could lead to a new range of antimalarial drugs whose structure and mode of action are different from those currently in use. Hemozoin formation, fatty acid biosynthesis, isoprenoid biosynthesis, heme biosynthesis, mitochondrial electron transport system and transport systems are among the potential drug targets for the development of new antimalarial pharmacophores. Pentose Phosphate Pathway (PPP) is one of the important pathway in malarial parasite P. falciparum. This pathway is an important metabolic pathway as it produces reducing power in the form of NADPH necessary for most vital antioxidant defense system and pentose sugar necessary for nucleic acid synthesis. Therefore, it would be interesting to study the pathway with a view to explore its chemotherapeutic potential. Transketolase is one of the regulatory enzymes in the PPP which is mainly responsible for the supply of pentose sugar required in nucleotide synthesis which is essential for the fast replication of the malarial parasite. For this, transketolase operates non-oxidative arm of PPP in reverse direction to utilize F6P and G3P produced by glycolytic pathway. It was reported by various workers that more than 80% of the parasite nucleic acid is derived directly or indirectly from non-oxidative part of PPP. In addition to important role in nucleic acid synthesis, transketolase, in absence of transaldolase, is likely to be responsible for generation of erythrose-4-phosphate from F6P and G3P. Erythrose-4-phosphate is a key metabolite funneled into shikimate pathway for the production of aromatic precursor chrosimate which is further metabolized into several aromatic compounds including folate. A search was launched to locate transketolase gene (PfTk) in P. falciparum genome. Attempts were made to amplify the Tk gene by PCR from P. falciparum genome with the objectives of cloning and expression of Tk gene. Successful heterologous expression in E. coli was followed by purification of recombinant protein. Enzyme assay was developed and standardized for the validation of transketolase activity of the purified protein. The purified protein was biochemically and biophysically characterized. Later, specific inhibitors for PfTk were identified and their possible in vivo antimalarial activity was evaluated. Simultaneously, efforts were made to study its localization and model structure construction. Chapter 1 deals with the cloning, expression and characterization of putative transketolase gene from P. falciparum. The strategy applied for PCR amplification of complete open reading frame of putative transketolase gene from genomic DNA of P. falciparum has been described. Evidences have been provided for successful cloning of PCR amplified fragment into E. coli expression vectors. Optimization of conditions for maximum expression of cloned gene in soluble form has been presented. Purification of recombinant protein was attempted by affinity chromatography using nickel nitrilotriacetic acid agarose resin and ammonium sulphate precipitation. Description of purification was followed by the biochemical characterization of enzyme. Details have been presented in support of enzymatic activity of the purified protein and was completed in terms of kinetic parameters. Chapter 2 highlights the localization of PfTk in the parasite. Purified recombinant protein was used to raise polyclonal antibodies against it and specificity of the raised antibodies was evaluated using enzyme linked immunosorbant assay (ELISA) and Western blotting. Western immunoblotting and confocal microscopic evidences using these polyclonal antibodies have been presented to illustrate the localization of transketolase in the P. falciparum. Chapter 3 deals with the effect of known transketolase inhibitors on the activity of PfTk. Using the standardized assay system, two potent inhibitors of purified recombinant PfTk were identified. To evaluate the anti-malarial activity of these inhibitors in vivo studies were conducted. Chapter 4 is concerned with the construction of homology model of PfTk and to search a new pharmacophore from CDRI’s 3D compound library. In order to explore the structural similarity of PfTk with existing transketolase structure, a homology model of PfTk was constructed. Circular dichroism spectropolarimetric evidences have been presented to validate the three dimensional model. Using the homology model of PfTk, binding property of both the identified inhibitors in PfTk were optimized on the basis of prior information available for template. The binding property of these inhibitors were used to perform a pharmacophore search of CDRI’s 3D compound database to identify new effective PfTk inhibitor.