&lt;i&gt;In-silico&lt;/i&gt; Rational Protein Engineering and Design Approach to Improve Thermostability of a Haloalkane Dehalogenase Enzyme

Raghunath Satpathy,Jagnyeswar Ratha,V Badireenath Konkimalla

doi:10.3844/ajbsp.2015.34.46

Abstract

Thermostability of enzymes is a major prerequisite for use in industrial enzymology. There are as such no simple general principles for achieving thermostability in case of enzymes as many factors are required to fulfil for different enzymes. The present study describes computational methods to design thermostable haloalkane dehalogenase enzyme using the crystal structure available Protein Data Bank (PDB ID: 1EDE). In in silico design strategy rule-based approaches such as disulfide bond geometry, new hydrophobic pocket design, new salt bridge construction and multiple mutations (combination of the above approaches) were introduced to the original enzyme. After each design strategy the functional effect was confirmed in terms of enzyme substrate binding by molecular docking using Autodock vina tool. Best design strategy was evaluated by comparative molecular dynamics simulation applying simulated annealing method at 8 ns using GROMACS tool. The surface hydrophobicity which is the key factor for thermostability in haloalkane dehalogenase was obtained from the simulation result. Upon optimizing the parameters, thermostability of mutant enzyme under consideration was also confirmed by the 5 ns molecular dynamics simulation at 400, 500 and 600 K.

Highlights

Haloalkane dehalogenase enzymes can be used as a potential catalyst for biodegradation of a number of halogenated compounds that are environmentally toxic expelled as industrial by-products (Fetzner and Lingens, 1994; Koudelakova et al, 2013)
In the current work a computational effort has been made to improve the thermostability of a haloalkane dehalogenase enzyme, thereby understanding the thermostability mechanism of the enzymes
During MD simulation, various properties such as the average values of the Solvent-Accessible Surface Area (SASA) for both hydrophilic and hydrophobic, the Radius of Gyration (RG), the number of inter and intraprotein hydrogen bonds and the number of salt bridges were computed as a function of time

Summary

Introduction

Haloalkane dehalogenase enzymes can be used as a potential catalyst for biodegradation of a number of halogenated compounds that are environmentally toxic expelled as industrial by-products (Fetzner and Lingens, 1994; Koudelakova et al, 2013). The catalytic reaction mechanism of the haloalkane dehalogenase enzymes (EC 3.8.1.5) involves the conversion of 1-haloalkane to a primary alcohol and halide ions following a hydrolysis reaction (Wackett, 1994; Prokop et al, 2003). These enzymes belong to alpha/beta hydrolase superfamily that acts on halide bonds present in carbonhalide compounds. The current in silico approaches in the protein engineering and design to enhance the thermostability in enzymes have been

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