Study On The Function Of Active-site Architecture Of Major Subfamilies Of Glycoside Hydrolase GH5

Lignocellulose is the most abundant renewable resource on the earth,and its main components include cellulose and various hemicelluloses such as mannan and xylan.Through the catalytic degradation by glycoside hydrolases,these polysaccharide components can be hydrolyzed into soluble sugars,which can be converted into biofuels and various chemical products through biorefinery.This provides a chance to solve the global problems,such as environmental pollution and energy shortage.Obviously,obtaining different kinds of glycoside hydrolases suitable for a specific industrial environment is the key to reducing industrial production costs and accelerating lignocellulose conversion.Although there have been many successful precedents for the design of glycoside hydrolases,the understanding of their active-site architecture and the molecular mechanism of the catalytic process are not yet deep.Based on structural bioinformatics method,this thesis systematically analyzed the sequence,structure and molecular dynamics of cellulase and mannanase from major subfamilies of GH5,and explored the active-site key amino acid residues related to the substrate binding specificity.In addition,using alanine scanning and functional analysis of key residues in the TfCel5A active site,the different function of key residues at different subsites in enzyme catalysis were preliminarily elucidated.The main contents of this thesis are as follows:1.Through structural bioinformatics analysis of enzymes from six major subfamilies in the glycoside hydrolase GH5 family,the main similarities and differences between different subfamily enzymes were elucidated.The sequence,structure and molecular dynamics analysis of the major subfamilies in the GH5 family revealed that the major differences in enzymes from different subfamilies exist in the eight loops that make up the active-site architecture.Comparing the active site sequence profiles of different subfamilies,the residues at negative subsites of the GH5 family were the most conserved.three residues at the-1 subsite were completely conserved throughout the GH5 family,and a tryptophan at the-2 subsite was also completely conserved,these residues may be closely related to catalytic cleavage;some residues are conserved or relatively conserved within a single subfamily,and these residues may be related to functional evolution between different subfamilies.2.Through structural analysis and biochemical determination of GH5₂ cellulase and GH5₈ mannanase,it was found that the substrate recognition mechanisms of the key residues at-1 and-2 subsites were different..Due to the different degrees of distortion of the-1 subsite sugar ring of cellulose and mannan substrate,there are differences in the spatial position of 03 at this subsite.However,at the-2 subsite,the difference between cellulose and mannan lies in the spatial location of 02.Correspondingly,structural alignment revealed that at the-1 subsite,the conserved histidine and asparagine of the two subfamilies played the role of substrate recognition by interacting with-1O3,while at the-2 subsite,the relatively conserved glutamate of the GH5₂ subfamily plays a role in substrate recognition through interaction with-202.3.Through the alanine scanning of all active-site amino acid residues of TfCel5A and biochemical determination,the different roles of active-site residues in the enzymatic process were revealed.Alanine mutations,binding constants Ka,and enzyme activity assays were performed on 22 key amino acids in the TfCel5A active site,and the functions of key residues in enzyme catalysis were analyzed.Ka assay results showed that the negative subsite residues are critical in substrate binding,where the conserved tryptophan of the-2 subsite and the relatively conserved charged-residue pair of-2/-3 subsites make up hydrogen bond network with substrates in the-2/-3 subsites,which plays an important role in the initial binding of the substrates.Enzyme activity assays showed that the-2 to +1 subsite residues are critical iin catalytic cleavage.iin which the conserved histidine and tyrosine interacting with-102 stabilize the nucleophile and maintain its charged state,while the conserved histidine and asparagine interacting with-103 assist in the distortion of the-1 subsite saccharide ring through interaction with the substrate,which requires a large movement of loop3;in addition,two conserved tryptophanes at the-2 and +1 subsites are also involved in the substrate deformation process;based on the high degree of conservation of the relevant residues in the GH5 family,their important roles in catalytic cleavage are consistent throughout the GH5 family.