Study On The Cysteine Protease Of Hyperthermophilic Archaea Pyrococcus Horikoshii OT3

The thermophilic enzyme from archaea Pyrococcus horikoshii OT3 (PhpI) is characterized by its unique high catalytic activity and stability, there is growing concern about the research of structure-function relationship and regulation mechanism of thermophilic enzyme. The study not only enriches protein engineering theory of the field, but also laid the foundation for industrialization.Based on the cysteine protease (PhpI), which is highly expressed in E. coli BL21 (DE3). Phpl exits in the form of oligomer, the basic uint is a monomer that is composed ofα/βhydrolase domain with 166 residues, and trimer above has activity. The active center is located on the interface of adjacent subunits, the catalytic triad formed by (A) C100-(A) H101-(C) E474. The research on its dodecamer found that the optimum temperature and pH are 85℃and pH 8.0. It has aminopeptidase and endopeptidase activity. The optimal substrate is R-AMC. Thermostability, reaction kinetics, enzyme activity and other influence factors have been characterized, the results show that the enzyme was the first allosteric protease discovered in DJ-1 superfamily. Random mutation library is conducted with Error Prone-PCR, through screening about 5000 clones, the mutant whose protease activity is increased by about 2-19 times is obtained. Gene sequencing shows that:the specificity of E12T increases about 3.8 times than wild type, K43C increases by 5.8 times, Y120P increases by 19 times. Combined with crystal structure. sequence alignment and bioinformatics method for analysis, the results show that the Y120 site is highly conserved in DJ-1 family, it not only combines with the nucleophilic residue C100 by hydrogen bond, but also uses benzene by P-πeffect to stabilize H101 which is another number of catalytic triad, therefore it expands the active sites. Substrate docking structure shows the 120 site just in the entrance of the substrate binding cavity, decreasing the accessibility of substrate entrance. Thus, the site affects the catalysis and substance binding. Crystal structure analysis suggests that there is an anion binding between R113 and N129 situated in the interface. two salt bridges formed by the guanidine group of R113 and sulfuric acid root ion, hydrogen bonds formed by water and N129. The carboxyl oxygen of Y120 and 1123 shape hydrogen bond, and there are hydrogen bonds among 1123 and D125. D126. The site-directed mutagenesis are carried out on 113.129 site on the basis of WT and 120P, the result shows that anion binding site involved in allosteric regulation, so the 120 site is also involved in allosteric regulation. From the above.120 site is the linchpin which can affect allosteric regulation, catalytic regulation and substrate combination.By measuring WT and mutants allosteric parameters and conducting the computer model of enzyme-substrate interaction, this paper explores the impact of Y120 site to Phpl catalysis, substrate binding and allosteric regulation deeply. Identifies the Y120 site is landmark site in the evolution of DJ-1 superfamily.