Study On Salt Tolerance Mechanism Of Leucine Dehydrogenase Based On Molecular Dynamics Simulation

Halophilic enzymes from halophilic microorganisms can not only survive in high salt environments,but also maintain good stability and activity.In this paper,the leucine dehydrogenase(psLeuDH)derived from the marine Pseudoalteromonas spongiae strain was used as the research system,and the enzyme was designed rationally.The molecular dynamics(MD)simulation method was used to simulate the dynamic changes of the enzyme during the simulation of different salt concentrations and try to explain the salt haloadaptation of the enzyme.First,the study on salt tolerance of psLeuDH was studied and the results showed that psLeuDH was extremely halotolerance enzyme,so psLeuDH can be used as research system.The three-dimensional structure of psLeuDH protein was constructed by homology modeling method with EasyModeller4.0 and the protein structure was analyzed.The enzyme was rationally designed to change the salt tolerance of the enzyme on the basis of previous studies.The mutants M1,M2 and M3 and M4 were obtained with the purpose of reducing the enzyme-dependent and improving salt tolerance by mutating the residues on the surface of the protein.The mutant protein three-dimensional structure was also obtained by homology modeling method with Easymodeller 4.0.The results of salt tolerance experiments of mutants Ml and M2 showed that the salt dependence decreased by reduecing acidity of the surface of psLeuDH,while the results of M3 showed that the salt tolerance was greatly enhanced by increasing the content of surface acidic amino aicds.The experiment showed that M4 was deactivated after mutation.For more direct observation of how the high salt environment affects the structure and behavior of the enzyme,molecular dynamics simulation of psLeuDH,M1,M2,M3 at 298 K and salt concentration of 0,2,3 M was carried out by NAMD software.The protein which has higher salt tolerance showed better stability in the high salt environment,which is consistent with the experimental results.The results of radius of gyration showed that the protein has different degrees of shrinkage under its appropriate salt concentration,which leads to the shortening of the spatial distance of the active site,While this change can accelerate the transfer of the proton and enhance the catalytic efficiency.With the increase of salt concentration,the decrease of SASA value of psLeuDH,Ml and M2 indicated that the affinity between enzyme and solvent was decreased which decreased the enzyme solubility.In addition,the results of SASA and surface ion distribution showed that a stable hydration network was formed by the side chains of the acidic residues on the surface of the protein with sodium ions and water molecules.After the mutation,the hydration network stability of M1 and M2 was destructed.On the contrary,M3 formed a more stable hydration layer which greatly enhanced the enzyme stability.The electrostatic interaction between chloride ion and side chains of Lys68 and Lys80 at different salt concentrations was observed at the active sites and the electrostatic interaction differs at different enzyme systems.This may result in the differences acitivity of enzyme at different salt concentrations.