Date of Award
Doctor of Philosophy (PhD)
Biotechnology Science and Engineering
Joseph D. Ng
Luis R. Cruz-Vera
Pyrophosphates., Enzymes., Bioinformatics.
Family I and Family II inorganic pyrophosphatases (IPPases) are soluble, metal-dependent enzymes that catalyze the hydrolysis of inorganic pyrophosphate into two orthophosphate molecules, and they affect several major metabolic pathways. IPPases are essential for life in both prokaryotes and eukaryotes, and they are potential drug targets. Family I and Family II IPPases were studied in order to provide platform for future drug-designing studies. ResCon, a standalone, cross-OS platform software suite with GUI, was developed in Python. It aids in extraction of homologous sequences from a large dataset containing multiple protein families, and then analyzes conservation of residues only at positions of interest, based on their multiple sequence alignment. From sequences retrieved from NCBI RefSeq Protein database, Family I-P (prokaryotic origin), I-E (eukaryotic origin) and II IPPase sequences were successfully isolated using ResCon. Active site residues are well conserved in Family II IPPases (98.7 %) compared to that in Family I-P (95.2 %) and I-E (86.3 %) IPPases. Certain active site residues were more likely to be affected than the others. For example, active site residue Asp67 is highly susceptible for mutation in Family I-P IPPases, but its equivalent residue in Family I-E IPPases is not likely to be affected. 149 Species, including 10 eukaryotes, 31 bacterial pathogens and a pathogenic strain of Escherichia coli, were found to contain both Family I and II IPPases. Besides active site residues, significant residue positions are well conserved with ≥ 91 % identity. The residues interior to the IPPase structures are more conserved than those exposed to the surface. Salmonella typhi is a bacterial pathogen, and its Family I IPPase is a model target for structure-based drug design. S. typhi IPPase crystals were obtained, and they diffracted to a maximum resolution of 1.95 Å and belonged to space group I23 with unit cell parameters a, b, c = 142.0 Å with all angles to be 90o. The IPPase structure was determined using molecular replacement revealing a homohexameric quaternary structure similar to that of E. coli IPPase. In examining a representable eukaryotic IPPase, the Human IPPase gene was synthesized, expressed in an E. coli recombinant system, purified and underwent crystallization trials. The recombinant Human IPPase had optimal pyrophosphate hydrolysis activity at 37 °C and pH 7 in presence of magnesium ions. Crystallization of Human IPPase yielded poorly diffracting crystals. Despite various attempts to improve crystallization, by using thermal shift assay, heterogeneous nucleation, in situ proteolysis and rational protein engineering, crystallization quality could not be improved. By combining evolutionary residue conservation data to crystal structure of S. typhi IPPase, potential drug target sites were identified.
Gajapathy, Manavalan, "Molecular and functional analyses of inorganic pyrophosphatases" (2016). Dissertations. 89.