Study On The Function Of Interaction Network Of Key Residues Of Endocellulase

Cellulose is widely distributed in nature and is a kind of abundant renewable resource.Using cellulase to transform and utilize it efficiently has important strategic significance for the sustainable development and agricultural modernization in our country.Cellulose is the main component of lignocellulose,with a variety of different crystalline structure and twisted conformation,is the most abundant polysaccharide in nature.Cellulose degradation is mainly played by a variety of cellulases play a key role,of which endocellulases mainly catalyze the degradation of amorphous cellulose,cellulose degradation plays an important role.Different families of endocellulolytic enzymes have different active structures.The complex interaction networks formed between key residues,key residues and substrates,and the loop dynamics and stability of active site have an effect on the function of enzymes,so the exploration of the key residue interaction network and enzyme function will help us to understand more deeply the structural basis and catalytic commonality law of interaction between different family endocellulolytic enzymes and substrates.In this paper,sensitive fluorescence spectroscopy was used to accurately determine the changes in binding force caused by single hydroxyl mutations in the-2 key residues of the GH5,GH7,and GH12 families of endocellulase activity,and different key residues were explored.The size of the binding force contributed by the study explored the mechanism of interaction between key residues in the binding of substrates through molecular dynamics simulations,and further analyzed the relationship between interaction networks and loop dynamics on enzyme function.The main content of this article is as follows:1.Fluorescence spectrophotometric determination of binding capacity of Trichoderma reesei three endocellulase-2 subsite residuesUsing sensitive,convenient,and rapid fluorescence spectrometry to accurately determine the binding force changes caused by single hydroxyl mutations in the-2 subsites key residues of GH5,GH7,and GH12 family endocellulase,TrCel5A,TrCel7B,TrCel12A.The corresponding thermodynamic parameters were determined and the contribution of the critical residues of the-2 subsite to the substrate binding was determined.It was proved that the key residues of the-2 subsite play an important role in substrate recognition and binding.2.Simulation analysis of interaction network of three endocellase-2 subsite residuesMolecular dynamics simulations were used to analyze the interaction networks of the three endocellulases-2 subsite residues and found that there are complex interaction networks between the key residues that can stabilize the conformation of key residues and make the enzyme molecules accurately identify the binding of the corresponding substrate,and key residue mutations will often break the original interaction network,making the key residue conformation changes,thereby affecting the enzyme's binding force and catalytic efficiency.It is proved that the key residues in the active site of the enzyme play a role in the specific recognition,catalysis,and product release of the substrate through a complex interaction network of residues.The analysis of the kinetic simulation can indicate the direction of modification of the enzyme molecule.3.Functional analysis of endocellulase interaction network and loop dynamicsThe interaction network exists in the whole enzyme molecule.Besides the ability to affect the specific recognition of the substrate by the enzyme molecule,the dynamic and stability of the active center loop are also closely related to the interaction network between residues.Molecular dynamics simulations of the endonucleases of the GH5-5 subfamily revealed that the two loops of the active site-2 and 1 subsites have different dynamics and stability at different temperatures.The key residues in the loop are involved.The key interactions have a direct impact on the loop dynamics and temperature tolerance,which in turn affects the properties and functions of the enzyme molecules.