Effect of Point Mutations in the Clostridium thermocellum Cellulase on the Thermostability: A Study by Molecular Dynamic Simulation

The GH5 family of cellulases is one of the largest between 145 families of glycoside hydrolases[1]. This family has a high diversity in terms of substrate specificity[2]. For this reason, they are referred to be a suitable source for the degradation of plant biomass[3]. The CtCel5E cellulase of these bacteria have cellulase and xylanase activities, which also has optimum temperatures of 50 ° C and 60 ° C, and a pH of 5 and 6 respectively[4]. In this study, the enzyme crystal structure (4U3A) was first taken from the PDB database. Then, by using the SWISS-MODEL server, a model of this enzyme was made by homology modeling method. The quality of the model was confirmed with Qmean, ProSA-web and Ramachandran Plot tools. Also, the RMSD was calculated between the model and the crystal structure using Visual Molecular Dynamics software and SWISS-MODEL server was chosen as the appropriate tool for the modeling. The molecular dynamics simulation was then performed at 400 K for 50 nanoseconds to identify unstable regions of the protein. In the following, Molegro Virtual Docker software was used to find suitable sites for mutation. Mutated proteins were modeled by homology modelling method using the SWISS-MODEL server. E24D, I142E, W211Q point mutations were selected to stabilize unstable regions of protein and increase the thermal stability. Molecular dynamics simulation was then performed at 400 K for 50 nanoseconds for the mutated protein and the results of RMSD and RMSF between natural and mutated proteins were compared and analyzed. The results indicated a significant increase in protein stability, which showed 15% at RMSD and 22% at RMSF increasing in the protein stability

Keywords:

Clostridium thermocellum , Cellulase , Thermostability , Salt Bridge , MD Simulation