Study Of The Catalytic Mechanism Of GH10 Glycoside Hydrolase

Lignocellulose is not only a renewable biomass energy source but also a natural chemical raw material.Because of the abundant plant biomass productionon the earth,lignocellulose has important research value.The main chemical components of lignocellulose are cellulose and hemicellulose.The hydrolytic cleavage of these two polymeric sugars can produce smaller fragmented molecules,then used to produce biofuels and natural chemical products.Therefore finding effective methods to decompose these carbohydrate polymers is an important issue in molecular biology.In nature,the cleavage of glycosidic bonds in biomass is efficiently catalyzed by glycoside hydrolase（GH）.Glycosidic bonds are among the most stable chemical bonds in natural biopolymers.Compared with the uncatalyzed hydrolysis of glycosidic bonds,the hydrolysis efficiency of glycoside hydrolase was increased by 10¹⁷ times.Therefore,exploring the catalytic mechanism of glycoside hydrolase is of great significance for the study of biomass degradation.This paper investigated the catalytic mechanism of GH10 glycoside hydrolase,which can hydrolyze xylan in hemicellulose,by molecular dynamics simulations and computational methods such as QM/MM.The specific research contents are as follows:First,based on the crystal structure of GH10 glycoside hydrolase（PDB ID:1R87,4PUD）,the calculated initial model structure was constructed by superimposing the coordinates of the complex.Then the initial structure was optimized by molecular dynamics simulation and selected a representative structure for subsequent QM/MM calculations.In comparison of binding free energy of the complex before and after the catalytic reaction,it was found that the binding free energy of the system did not change significantly after the sugar chain was broken.The binding mode and hydrogen bond occupancy of the active site xylan before and after catalysis were compared,and the effect of glycosidic bond breakage on the active site was analyzed.The key amino acids in the catalysis process were found by the difference between the binding free energy of amino acid residues within 3（?）of the active site xylan before and after catalysis:ASN59,ASN158,GLU159,GLN234,TRP316.Finally,the QM/MM method was used to calculate the catalysis of GH10 glycoside hydrolase.In the xylan chain cleavage reaction pathway,the reaction relies on two displacement reactions to break the glycosidic bond.The transition states and intermediates in the reaction process were found,and the interaction of the active site xylan and the electron transfer in the transition state were analyzed.The changes insugar chains during the reaction were measured,and it was found that when GLU265and water molecules carried out a nucleophilic attack on the 4XB376@C1 atom,the pyranose ring skeleton was twisted into a semi-chair structure to promote the reaction,and shortens the distance between the C1-O5 atoms respectivelyto proceeds reaction smoothly.This paper provides a theoretical basis for the catalytic mechanism of GH10 glycoside hydrolase,thus providing a reference for improving the enzymatic engineering of the glycoside hydrolase family.