Design of enzyme with new substrate specificity using computational methods

Abstract

Enzymes are widely used in many chemical synthesis and medicines. The use of enzymes in industrial production is newly emphasized for environment-friendly and sustainable development. For their industrial use, enzymes are needed improved characteristics such as specificity, stability, enantio-selectivity and high-level expression. Enzymes from nature have been modified to have improved properties by rational engineering or directed evolution method. In recent years, computational methods for the design of enzyme have been available by the wealth of structural data and increasing computing power. In this research, computational design method was developed for the improved substrate specificity of hydantoinase. Major concern in the design was how to set biological constraints in terms of computable terms. From the main idea of computing minimum energy structure, many modified terms and constraints were applied to increase accuracy while reducing calculation time. Basic computation system is composed of energy calculation with Amber force field, various calculation parameters, rotamer library and MC optimization. Enzyme-ligand docking modes were prepared before energy calculation. Several hundred docking modes were selected while target amino acids positions were mutated to alanine and while distance between catalytic carbon of hydantoin and nucleophilic water molecule was conserved. Target residues were selected based on structural investigation, mechanistic knowledge and preliminary experiments. Initially two kinds of approach were applied, which were fixed model and dynamic model. The calculation parameters were optimized separately according to each model. After the design calculations were performed, experimental candidates were selected and the activities of them were analyzed. Following the first design procedure, the parameters of the scoring function were modified to fit new experimental rank order. As the rounds proceeded, the specific activity of be...