Catalytic Property Modification Of Halohydrin Dehalogenase HheB_GP1 Inspired By Structural Analysis

Halohydrin dehalogenases not only catalyze the dehalogenation of vicinal halo alcohols to the corresponding epoxides through an intramolecular nucleophilic substitution mechanism,but also catalyze the ring-opening reaction of epoxides mediated by nucleophiles.Halohydrin dehalogenases participate in the biodegradation of organic halide contamination in the environment in nature,and play an important role in industrial production such as the synthesis of optically pure epoxides,?-substituted alcohols and chiral drug precursors.Previous studies have shown that amino acid 178 in Hhe A_AD2,a member of the halohydrin dehalogenase family,plays an important role in regulating the enantioselectivity of the enzyme;the modification of another member of the family,Hhe C_AD1,found that amino acid 176 has a similar regulation.The crystal structures of the two were superimposed,and it was found that the above two sites were in the same spatial position of the crystal structure.In addition,the mutations at sites 136 and 141 of Hhe A_AD2 and 134 and 139 of Hhe C_AD1 both improved the enantioselectivity of the enzyme,and these sites correspond to each other in the spatial structure,suggesting that the above sites are important for the halohydrin dehalogenase family.In this thesis,another member of the halohydrin dehalogenase family Hhe B_GP1 was used as the research object to systematically analyze the effect of the amino acid residues corresponding to the above three spatial positions on the enantioselectivity of Hhe B_GP1.Firstly,homology modeling and structural comparison of Hhe B_GP1 were performed to find the corresponding sites,namely T120,H125 and H162 sites.Two saturated mutant libraries,T120X/H125X and H162X,were constructed by the"small-intelligent"method,and 1400 and 150 single clones were screened,respectively,and two dominant mutants,T120L/H125Q and H162G,were obtained.The catalytic properties of the mutants showed that the catalytic activity of T120L/H125Q towards 1,3-DCP was 3.6 times higher than that of the wild type,and the enantioselectivity towards rac-2-CPE was reversed from E_S=7.1 of WT to E_R=46.3;while the catalytic activity of H162G towards 1,3-DCP is only 13%of that of WT,the catalytic activity towards rac-2-CPE is 67 times higher than that of WT,and the enantioselectivity increases from E_S=7.1 to E_S>200.On this basis,the dominant mutants were used as templates,and the three sites were superimposed through the second round of saturation mutation studies to expect further improvement of catalytic properties.The results showed that the six dominant mutants screened all contained the H162G mutation,and the enantioselectivity E values for S-2-CPE were all greater than 200.Taken together the above results show that the amino acids at these three spatial positions,especially the 162 position(corresponding to the other two enzymes 176-Hhe C_AD1;178-Hhe A_AD2),also have important regulatory effects on the enantioselectivity of Hhe B_GP1 like Hhe A_AD2 and Hhe C_AD1.So the hypothesis is verified that the amino acids in the three steric positions have improtant effect for enantioselectivity in halohydrin dehalogenases.The reasons for the improved catalytic properties of the mutants were analyzed by molecular docking,which laid the foundation for further study of halohydrin dehalogenases.In addition,this thesis further modified the Hhe B_GP1.It is hoped to obtain a dominant recombinant that fuses the advantages of the three enzymes Hhe A_AD2,Hhe B_GP1,and Hhe C_AD1 by replacing the catalytic core regions of the above three halohydrin dehalogenase family members by random recombination.