Molecular Dynamics Simulation-based Study On The Catalytic Mechanism And Design Of Thermally Stable Mutants Of Nattokinase

In recent years,there has been a significant increase in the incidence of cardiovascular disease worldwide.Finding effective and safe thrombolytic agents has become one of the directions in the field of cardiovascular disease research.Nattokinase,a potent thrombolytic agent,is considered a new potential thrombolytic drug.However,the energy and structural changes during the catalytic process of nattokinase are not yet clear,and its thermal stability is poor,which hinders its application in the pharmaceutical field.In protein engineering,molecular dynamics（MD）simulation can be used to explore the interactions and movements between molecules in the system.With the improvement of computer performance and calculation methods,the quantum mechanics/molecular mechanics（QM/MM）combined method has been widely used to establish enzyme catalytic reaction mechanism models.To investigate the detailed information of the catalytic process of nattokinase,this study performed a QM/MM umbrella sampling simulation on the nattokinase-tetrapeptide substrate complex.The binding free energy was calculated using the MM/GBSA method,and the contribution of each amino acid residue to the binding energy was decomposed.The simulation results showed that in the catalytic reaction of nattokinase,the hydroxyl proton in Ser²²¹ was transferred to the nitrogen atom at position 3 of the imidazole ring of His⁶⁴,enhancing the nucleophilicity of Ser²²¹ to form an intermediate with a system energy of 3.9 kcal/mol.The hydrogen bond between His⁶⁴ and Asp³² stabilized His64.Asn¹⁵⁵,as the oxygen anion hole of nattokinase,formed a hydrogen bond with the oxygen in the substrate peptide bond in the transition state.To screen and construct nattokinase mutants with higher thermostability,this study used a free energy-based method to obtain potential nattokinase mutants with improved thermal stability.The mutants were then screened using molecular dynamics simulations and validated with relevant experimental analyses,resulting in three active mutants,E156F,Q206L,and Y256P,which were able to enhance the thermostability of nattokinase.Molecular dynamics simulations were then performed on these three mutants at higher temperatures to analyze the inherent mechanism of thermostability improvement.The results showed that mutating hydrophilic amino acids located on or near the enzyme surface to hydrophobic amino acids can enhance the thermostability of nattokinase,and factors such as lower root-mean-square deviation（RMSD）,lower radius of gyration（Rg）,decreased solvent-accessible surface area（SASA）,and increased rigid amino acid residues near the active site are all contributors to the enhancement of nattokinase thermostability.In summary,the following innovative work has been done in this paper:（1）The energy and structure change information of the transition state of nattokinase catalyzed reaction was clarified.（2）Discover nattokinase mutants that can improve thermostability and propose the internal mechanism of the mutants’improved thermostability.