Structure and Dynamics Studies on SAICAR Synthetase from Pyrococcus horikoshii OT3

Understanding the structural details of a biological macromolecule is highly essential for a clear interpretation of its functional roles. Structural biology evolved over a period of time has been able to explain various cellular processes which has consequently lead to the discovery of several effective drugs. X-ray crystallography is one of the powerful tools in structural biology, contributing to ∼88% of the three-dimensional structures deposited in the Protein Data Bank (PDB). A single experiment can however provide only one of the numerous conformational states a macromolecule can exhibit. This setback is overcome by classical mechanics based molecular dynamics simulation technique developed based on various force fields that can generate many possible conformational states a macromolecule can take up. This thesis describes the use of both X-ray crystallography and molecular dynamics simulation techniques to elucidate the structural and dynamic aspects of a thermostable enzyme from a hyperthermophilic organism focusing on its thermostability and catalytic mechanism. Nucleotides have significantly numerous roles in the cell, forming the basis of an organ-ism’s genome, providing cellular energy, assisting as coenzymes in many enzyme catalyzed reactions, acting as secondary messengers etc. These biologically important chemical entities are classified into purines and pyrimidines. Biochemical synthesis of either purine or pyrimidine nucleotides are accomplished either by de novo or salvage pathway. SAICAR synthetase is one of the enzymes involved in de novo purine biosynthesis pathway. It catalyzes the seventh (in humans and higher eukaryotes) or eighth step (in bacteria and fungi) of de novo purine biosynthesis pathway. The enzyme is an ATP dependent ligase which forms C-N bond between 5-Amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate (CAIR) and L-aspartate (ASP), in the presence of magnesium, resulting in the formation of 5-Amino-4-imidazole-N-succinocarboxamide ribonucleotide (SAICAR). An overview of the structural and biochemical aspects of SAICAR synthetase along with an introduction to the structural and dynamic basis of protein thermostability is described in chapter-1. The work described in this thesis starts with the clone of SAICAR synthetase. A series of procedures involving protein expression and its subsequent purification results in a pure protein which is crystallized by underoil microbatch crystallization method using appropriate conditions. X-ray diffraction data of the protein crystals are collected using Cu-Kα radiation (1.5417 A) as a source on MAR Research image plate detectors. Diffraction data are processed using IMOSFLM, scaled using SCALA. Molecular replacement method is used for solving the structures using PHASER. Refinement of the structures are performed using REFMAC. Model building of the structures are carried out using COOT. Structures are validated using RCSB validation tools. Simulations of the structures are carried out using a popular open source program, GROMACS. A…