STUDIES OF THERAPEUTIC PROTEIN TARGET: ORNITHINE DECARBOXYLASE FROM ENTAMOEBA HISTOLYTICA

Abstract

Entamoeba histolytica is a single-celled parasitic protozoan responsible for causing amoebiasis. The parasite infects liver and intestine, which may cause mild diarrhea and life threatening abscissions. E. histolytica is responsible for over 50 million infections in tropical and. temperate regions, and nearly 100,000 deaths worldwide each year. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate homodimer and active site is located at the dimer interface. However, partially purified ODC from E. histolytica. (EhODC) is reported to exist in a pentameric state. The drug a-difluoromethylornithine (DFMO), a potent substrate analogue inhibitor of ODC, is widely used for the treatment of various diseases including Trypanosoma brucei infections. However, this drug is ineffective against E. histolytica. To analyze such differences we compared the sequence of EhODC enzyme with ODCs from wide range of organisms, which demonstrated the conservation of the sequence of EhODC at the active site and dimer interface. We re-investigated the oligomerization state of EhODC to identify and characterize the active oligomeric form of EhODC. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in the dimeric form. Further, computational model of EhODC dimer was generated. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value -0.327 nm. We have generated different mutants by site-directed mutagenesis to determine that the dimeric state is the active form of EhODC and to study the role of residues at the dimer interface. These studies signifies that active EhODC is a functional homodimer where two active site pockets form at the interface due to some long and short range of interactions at the interface. The residues important for dimer formation present at the interface are responsible for keeping two monomers in close . proximity. Disruption of dimer disfigured the active site pocket which results in the inactivation of EhODC enzyme. Further, realizing the importance of structure based drug designing, we determined the crystal structure of EhODC at 2.8 A resolution. Structure was solved by molecular replacement method. The enzyme forms orthorhombic crystal exhibiting P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 A. Structure, sequence and phylogenetic studies of EhODC reveal its evolutionary relationship with homologs of active and inactive ODC as well as structural modifications rendering its resistance towards DFMO. This thesis is divided into five chapters and covers the characterization of therapeutically important protein: ornithine decarboxylase from Entamoeba histolytica, which is Pyridoxal 5' phosphate dependent enzyme of fold III group IV decarboxylase. Enzyme has been characterized on the basis of biochemical, mutational, in silico and 3D crystal structure analysis. Chapter 1 Reviews the literature; describes the life cycle and the genome organization of Entamoeba histolytica, metabolic pathways; polyamine biosynthetic pathway; role of polyamines in various life processes; regulation of polyamine biosynthesis by ornithine decarboxylase (ODC); pyridoxal 5' phosphate dependent enzymes; classification of ODC; reaction mechanism and Inhibition of ODC; regulation of ODC by antizyme and antizyme inhibitor; structural and mutational studies of ODC. Chapter 2 Describes the over-expression, purification and biochemical characterization of recombinant EhODC. The expression of EhODC enzyme was done in E. coli BL21 (DE3) cells transformed with pET30a-EhODC construct having enterokinase cleavage site. Protein was purified to homogeneity by two step procedure that employed metal ion affinity chromatography followed by gel filtration chromatography. Protein was found to be expressed best at 18 °C for 14 hours. Enzymatic activity of EhODC was determined spectrophotometrically by product (putrescine) detection colorimetric assay and found to be active in the presence of L-ornithine. Oligomeric state determination was done by various methods. 1) Crosslinking experiment was performed using 40 μl glutaraldehyde solution (12.5 % v/v) acidified with 1 μl 5 N HCI. The sample was analysed on 12 % SDS-PAGE and found to be crosslinked to form dimer of —90 kDa. 2) The purified protein was applied onto a HiLoad 16/60 Superdex 200 gel filtration column and compared with elution volume of standard protein marker. It was confirmed to be a dimer of —90 kDa. 3) Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI/TOF MS) was performed. The mass spectrometer revealed two peaks corresponding to molecular weight —45 kDa and -90 kDa. 4) The effect of urea and NaCl on oligomeric state of EhODC was determined at different concentrations (2 M or 4 M), and analyzed by loading on Hi-load 16/60 superdex 200 gel filtration column. The concentration of urea and NaC1 were estimated to have adverse effect on the oligomeric state of protein and increase in concentration result in the disruption of the dimer to monomer. For secondary structure determination circular dichroism (CD) of protein was performed. The secondary structure content of EhODC was comparable to other well characterized ODC. Chapter 3 Describes the bioinformatics, computational analysis and molecular modeling of EhODC. Three-dimensional (3D) homology model of EhODC homodimer was generated by comparative modeling using MODELLER 9v8. Molecular dynamics (MD) simulation of dimeric model. of EhODC was performed using GROMACS (v 4.5.4) package. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulation was performed and the dimeric structure was found to be very stable with RMSD value —0.327 nm. Chapter 4 Describes the site directed mutagenesis studies of EhODC. Residues playing critical role at the active site and at dimer interface were studied in detail. The residues present at the interface and responsible for dimer stability and active site residues responsible for catalysis were mutated. Expression, purification and enzymatic activity of different mutants were done using the same protocol as used for purification of wild type EhODC. The results revealed that mutation of Lys57Ala or Cys334Ala completely abolishes enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Gly36lTyr and Lysl57Ala mutations at the dimer interface- were found to abolish the enzyme activity. Gel filtration chromatography analysis revealed that Gly36lTyr mutant was showing two peaks at the elution volume corresponding to dimer (-90 kDa) and monomer (--46 kDa). Further, Gly36lTyr-Lysl57Ala double mutant of EhODC was expressed at very high concentration of IPTG and found to very unstable during purification. Chapter 5 Describes the crystal structure of ornithine decarboxylase from Entamoeba histolytica. Cloning of the C-terminal truncated construct of EhODC was done in pET28c with TEV protease cleavage site. Expression, purification and enzymatic activity of 15AcEhODC was done using the same protocol as used for purification of wild type EhODC. The crystals were grown at 20 °C by sitting drop vapor diffusion method using 2 μ1 of protein solution mixed with 1 pl of reservoir solution containing 20 % PEG 3350 in 0.2 M LiCl solution maintained at pH 6.8 as provided in Hampton PEG ION screen. All diffraction data were collected using rotating anode X-ray source and solved by molecular replacement method. The orthorhombic crystal exhibiting P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 A. Sequence analysis revealed that EhODC is an evolutionary bridge between functional ornithine decarboxylase and nonfunctional antizyme inhibitor. Functional as well as evolutionary relation of EhODC with ODC homologs was established on the basis of sequence analysis, phylogeny and structure.