Please use this identifier to cite or link to this item: http://localhost:8081/xmlui/handle/123456789/14791
Title: STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF ARGINASE ENZYME FROM ENTAMOEBA HISTOLYTICA
Authors: Malik, Anjali
Keywords: Arginase Enzyme;Entamoeba Histolytica;Antiamoebic;Protozoan Diseases;Biosynthetic Pathway
Issue Date: 2019
Abstract: Entamoeba histolytica, a unicellular enteric dwelling human protozoan parasite which belongs to the family Entamoebidae, causes the disease amoebiasis, and infects around 50 million people in India, Latin America, Africa and South-east Asia each year. After excystation in the small intestine, the trophozoites of E. histolytica moves to the lumen of large intestine where they reside asymptomatically as a non-pathogenic commensal in case of most infected individuals, but in some cases due to some unidentified reasons these trophozoites become virulent and invasive, migrating to vital organs such as liver and lungs through the bloodstream causing life threatening diseases like amoebic colitis or amoebic abcession. Amoebiasis is manifested by onset of dirrahoea with 70 % patients finding blood in their stools, gradual weight loss, fatigue, abdominal pain & increased body temperature. Infection and transmission occurs through faecal-oral route either directly by person to person contact or indirectly through consumption of faecally contaminated food and water. Amoebiasis caused by E. histolytica results in 100,000 deaths each year making it one of the leading cause of death due to protozoan infection after malaria. Presently, a small number of antiamoebic drugs like chloroquine, nitazoxanide, metronidazole and tinidazole are used for the treatment of the disease. But these antiamoebic drugs are facing some problems due to the rapid development and spread of the drug resistant parasites. Additionally, some of these drugs have been found to have side effects. For instance, metronidazole is found to be tumorigenic and carcinogenic and nitazoxanide is also found to have side effects. These reports indicate that there is a serious requirement to identify and elucidate a potent metabolic pathway which could be used as therapeutic target for the development of antiprotozoan drug which can inhibit the growth of the parasite while having least impact on the health of the host. In last few decades, polyamine biosynthetic pathway has been therapeutically targeted for the development of potential drugs against parasitic protozoan diseases including leishmaniasis, giardiasis and African sleeping sickness. Polyamines are organic polycations which are essential for cellular processes like differentiation, signaling and apoptosis. They are also shown to participate in protein synthesis and cell cycle regulation, maintenance of chromatin structure and, stabilization of RNA molecule. Elevated concentrations of polyamines are found in the proliferating cells of parasitic protozoa, and repression of polyamine synthetic pathway by inhibiting the ornithine decarboxylase (ODC) of polyamine biosynthetic pathway II has been used in the clinical treatment of protozoan diseases like western African sleeping sickness caused by protozoan Trypanosoma brucei gambiense. Thus polyamine biosynthetic pathway has been considered as a potential drug target against the human pathogenic protozoans. Polyamines metabolism of E. histolytica is currently being exploited by targeting its ODC (EhODC) as it is the only enzyme of polyamine biosynthetic pathway of E. histolytica that has been structurally studied and characterized. But surprisingly, three dimensional structure and sequence analysis of EhODC has shown that the enzyme has evolved and turned out to be resistant to the substrate analogue difluoromethylornithine (DFMO). These findings necessitate the further exploration of the polyamine biosynthetic pathway of E. histolytica to discover novel drugs for antiamoebic therapeutics. The binuclear manganese dependent metalloenzyme arginase (EhArg) precedes ornithine decarboxylase in the polyamine biosynthetic pathway and may represent an alternate drug target for treatment of amoebiasis. Arginase catalyzes the catabolism of L-arginine to Lornithine and urea thereby maintaining the flux of L-ornithine for the polyamine biosynthetic pathway. In this process it depletes the local arginine concentration at the site of infection which affects the immune response by decreasing the concentration of nitric oxide generated by activated macrophages. Therefore, arginase plays a critical role not only in maintaining the polyamine biosynthetic pathway but also in evading the host immune response against the invading pathogen. The genome of higher eukaryotes code for more than one arginases whereas, genome of E. histolytica codes for single arginase enzyme. The full length mRNA of E. histolytica arginase (EhArg) encodes a 296 amino acid long protein with an amino acid sequence showing 36%, 35% and 33% identity to Bacillus caldovelox arginase (BcArg), Plasmodium falciparum arginase (PfArg) and human arginase (HArg) respectively. This study focuses on the structural, biophysical and biochemical characterization of arginase from Entamoeba histolytica. This thesis encompasses the cloning, expression and purification of EhArg followed by characterization of the oligomeric state in different solvent conditions, activity assay at different pH and salt conditions. The promiscuous nature of EhArg binding to different metal ions was determined by Surface Plasmon resonance (SPR) and further activity in presence of different metal ions was tested in biochemical assay. EhArg was co-crystallized with substrate analogue inhibitors and the product to determine its tertiary structure, active site chemistry and determinants involved in substrate binding. The knowledge of the molecular determinants involved in inhibitor and product binding was further utilized for III the screening and identification of novel non-amino acid based inhibitors and their inhibition potential were tested by calculating the IC50 values. The gene encoding for the arginase enzyme of E. histolytica was cloned in pET-28c vector harbouring N-terminal His-tag and TEV protease cleavage site. The cloned pET-28c vector containg the EhArg gene was then transformed into E.coli Rosetta (DE3) cells which act as an expression host. The expression of recombinant protein was performed at 18˚C and 0.2 mM of IPTG concentration. The over-expressed protein was then purified by immobilized metal ion affinity chromatography (IMAC) using Ni-NTA HisTrap affinity column. The protein obtained after affinity chromatography was further purified by size exclusion chromatography for separating the remaining impurities from the protein. The purified protein was used to perform several preliminary experiments to investigate the oligomerization pattern, secondary structure and stability prediction of the protein. Additionally, biophysical and biochemical characterization of EhArg was performed to explore the differential binding pattern of EhArg with metal ions other than the physiological activator of the enzyme. This study reports the metal binding kinetics of different divalent metal ions to purified EhArg through Surface Plasmon Resonance (SPR) and has revealed that EhArg is more promiscuous in metal binding than previously characterized arginases. The phylogenetic analysis of arginase has been performed that correlates the function and evolution of arginase in different organisms. Additionally, in the lack of the availability of the three dimensional structural information, the homology model of EhArg was built and molecular docking of two manganese ions in the active site was carried out to gain insights into the metal binding sites. ICP-MS experiments have been performed using purified EhArg samples. ICP-MS studies unveiled the fact that manganese was added to the recombinant protein inside the cell during its expression and production in bacteria. Moreover, its treatment with EDTA (Ethylenediaminetetraacetic acid) removed the surface exposed Mn+2 while the deeply embedded Mn+2 could not be extracted by EDTA treatment. Gel-filtration chromatography and activity assay have been performed in different enzyme reaction conditions for the enzymatic characterization of EhArg. As the three dimensional structure for arginase enzyme from E. histolytica is not available, crystallization of EhArg was performed for structural characterization of EhArg enzyme. The purified protein obtained after size exclusion chromatography was concentrated upto 10mg/ml and was used for crystallization and co-crystallization with the substrate analogue inhibitors and the product. Initial hits were obtained in the PEG ion screen from Hampton Research which was further improved by altering the protein concentration, IV temperature, concentration of salt and precipitant, pH and ratio of the protein to the reservoir buffer. Crystals of EhArg in complex with two substrate analogue inhibitors and product were obtained which diffracted at high resolution of 1.7Å, 2.0Å and 2.4Å respectively. Diffraction data were collected at the synchrotron beam line BM-14 at the European Synchrotron Radiation Facility (Grenoble, France) indexed and processed using XDS and scaled with SCALA. The structure was determined by molecular replacement with MOLREP using crystal structure of arginase from bacillus caldovelox (PDB ID: 1CEV) as the search model. The crystals exhibited I212121 symmetry with unit cell parameters a= 88.23, b= 97.2, c= 125.75. The detailed structural analysis of EhArg was performed followed by the comparison of EhArg structure with other structurally characterized arginases. Further, based on the structural analysis and previous literature survey Gly228 (Gly235 in humans) which lies adjacent to the residues involved in metal binding was mutated to arginine by site directed mutagenesis and the effect of this mutation was studied in determining the role of Gly228 in maintaining the active site architecture. The structural knowledge of EhArg paved way for the structure based screening and identification of the drug inhibitor molecules against the protein. Virtual screening of 1884 e- Drug3D molecules from e-LEA3D library was performed against the active site of EhArg, and the drugs showing the highest binding affinity were selected and docked into the active site of the EhArg protein using the AutoDock tool. The molecular dynamics simulation was performed to find the structure and dynamics changes occurring at the atomistic level in EhArg after the binding of the screened drug molecules. Molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) tool was used determine interaction free energy of protein-ligand complexes. The inhibitory potential of the screened drugs was determined by calculating the IC50 values using the biochemical assays. This study reports the detailed structural, biochemical and biophysical analysis of the protein along with identification of novel non-amino acid based inhibitors of arginase which can be used as the therapeutic modulators for the development of drugs against various human diseases by arginase targeted approach.
URI: http://localhost:8081/xmlui/handle/123456789/14791
Research Supervisor/ Guide: Tomar, Shailly
metadata.dc.type: Thesis
Appears in Collections:DOCTORAL THESES (Bio.)

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