Exploration Of Molecular Dynamics Behaviors In Cellulase Catalysis By Protein Dynameomics Method

Cellulose is the most abundant renewable resource on the earth.Cellulases can hydrolyze cellulose into soluble sugars which can be converted into biofuels and biochemicals by fermentation in bio-refinery industries.This provides a chance to solve the global problems,such as environmental pollution and energy shortage.Consequently,it becomes necessary to investigate the molecular mechanism underlying the cellulase catalysis.Despite of many successful works on the engineering of cellulases,the understanding of the relationship between protein structure,dynamics and function remains unclear.Through the combination of molecular dynamics simulations and other bioinformatics tools,we explore the key molecular mechanism associating with the catalytic properties of cellulase in different glycoside hydrolase families.Our results provide structural and conformational insights into enzyme catalysis,as well as strategical guidelines for engineering novel enzymes with optimized properties.The major contents in this dissertation include:1.Five GH12 cellulases with same folding topology but completely different thermostability are selected as our research model.The dynamics stability and structural stability determined the optimum temperature of enzyme catalysis cooperatively,which lays the foundation for comprehensive understanding of thermal inactivation of enzyme and the rational design of protein stability.Through constructing parallel molecular dynamics simulations at gradient temperatures,the structure and dynamics of enzyme are investigated upon temperature elevation.All cellulases could remain their structural integrity at room temperature.After raising temperature,principal component analysis shows that the collective motion of enzyme active site is perturbed,and the degree of perturbation is negatively correlated with the thermostability of GH12 cellulases.At higher temperatures,the N-terminuses of mesophilic cellulases unfold firstly,and theN-terminuses of middle-thermophilic and hyper-thermophilic enzymes unfold successively.Importantly,the internal interaction energy within N-terminus is positively correlated with the thermostability of GH12 cellulases,which suggests that N-terminus might determine the overall stability of GH12 cellulases.The thermal inactivation of enzyme includes reversible and irreversible steps,our results proven that the dynamics stability of active site and the structural stability of N-terminus are key factors for this process in GH12 family,these two factors independently and cooperatively determine the optimum temperature of enzyme catalysis.Furthermore,bioinformatics analysis reveals that thermophilic enzyme possesses more complicated interaction network which helps to stabilize its active sites as well as the whole structure.2.Through building the characterizing methods for enzyme active site conformational dynamics and exploring the major influential factors on active site conformations,the important roles of native active site conformational dynamics in cellulases catalysis are proved from different points.Enzyme catalysis requires the continuous adaption of active site conformation,so it becomes crucial to comprehensively describe its conformational dynamics for understanding the inherent mechanism of enzyme catalysis.One advantage of molecular dynamics simulations is the ability to record the trajectory of protein motion at atomistic level.Based on this,we can describe active site conformational dynamics at the level of overall structure,of secondary structure,and of amino acid residues.Besides,the key factors affecting conformational dynamics are investigated.Through analyzing the collective motions of active site,the dynamics perturbation of active site is found to mediate the reversible thermal inactivation of enzyme.Through analyzing the conformational dynamics of active site loops and residues,we proved that the open-close motion of active site and conformational selection of residues are necessary for binding substrate.Interestingly,we found that substrate binding exert repressive effect on the rebalance of active site conformational dynamics,which might cause the recession of cellulase activity in continuous-catalyzed system.In conclusion,these results reveal that the conformational selection during catalysis obey specific path,and both the perturbation to native conformational dynamics and to conformational selective path may damage enzyme activity.3.The preferences of conformational dynamics of active site residues at each position are discovered,and the optimization of active site conformational dynamics is proved to be another way for evolving protein function from evolutionary perspective.We selected twenty cellulases from GH5,GH7 and GH12 families as model,and characterized the conformations for hundreds of active site residues.By comparing the residue conformational distributions at corresponding position between different members in the same family,the preferences of conformational dynamics of active site residues at each position are determined.The results show that the residues at some positions have only one conformation but the residues at other positions have multiple prevalent conformations.Notably,it's generally applicable in a GH family,which implies the different roles of these two types of residues in catalysis.More importantly,the analysis of dynamics properties between different members discovers the significant evolutionary trend of active site conformational dynamics,which offers evolutionary evidence that the optimization of active site conformational dynamics might be another way for evolving protein function.4.Based on the analytic results of several GH families,the concept of protein dynameomics is proposed,and comparative molecular dynamics simulation systemsin terms of protein family are constructed.In the studies of protein thermal stability,the effector of protein dynamics and the evolution of protein dynamics in different GH families,using high-performance computer cluster,a big dataset containing the information of protein structural dynamics has been constructed.Molecular dynamics simulations for these enzymes offer more atomistic details for understanding the molecular mechanism underlies protein function.Confined to computer resource,only several enzymes are selected in our study,by quantifying the correlation between the dynamics features and functional properties of different enzymes within a family,we can determine whether the structural and dynamics features have real functional significance or not.And,the predictions are generally consistent with the experimental results.Based on these above results,we propose the concept of protein dynameomics,its basic meaning refers to the characterization and comparative analysis of the dynamic characteristics between different proteins and the coupling relationship between dynamics and function in specific protein family with the same structural topology.The concept of protein dynameomics enriches the methodology of protein and enzyme.In addition to application in cellulases,this method is also applicable to study the functional mechanism of other protein families.