Semi-rational Design Of Halohydrin Dehalogenase HheC For The Ring-opening Reaction Of Epoxide

Halohydrin dehalogenase can catalyze the dehalogenation of organic halides through the mechanism of intramolecular nucleophilic substitution,and it can also accept negatively charged nucleophiles to catalyze the reverse reaction,that is,the ring-opening reaction of epoxides,thereby halohydrins dehalogenase play an important role in the degradation of organic halides and the synthesis of various optically pureβ-substituted alcohols and epoxides.Among the halohydrin dehalogenases that have been discovered,because HheC from Agrobacterium radiobacter AD1 can efficiently catalyze short-chain substrates and show high enantioselectivity for R-type substrates,currently the substrate spectrum,ring-opening activity,enantioselectivity and practical industrial applications of the ring-opening reaction have been studied in detail.Most of these studies are based on wild-type HheC,but the catalytic properties of wild-type HheC such as activity and enantioselectivity cannot meet practical applications.However,there are only a few directed evolution studies on the ring-opening reaction of HheC.The main reason is the lack of a universal and efficient high-throughput screening method for enzyme’s activitiy for ring-opening reaction.Although two screening methods have been developed since 2015,but both have their own application limitations.Therefore,we constructed a universally applicable high-efficiency screening method based on the OH~-which the ring-opening reaction of epoxide produce can cause the changes of the p H of the system and the color of the p H indicator,and verified the sensitivity and stability of the method.The buffer concentration of the reaction system was optimized,and the interference of the nucleophile in the system was eliminated.Finally,the screening method was used the screen HheC error-prone PCR library to verify its effectiveness.On this basis,the combinatorial active-site saturation test,(CASTing)strategy was used to optimize the ring-opening activity of HheC.According to the crystal structure of the co-crystal of HheC and R-styrene oxide in the PDB protein database,the amino acid sites in the 4?range around the substrate R-styrene oxide are used as the core of this thesis.According to their spatial distance,these amino acid residues were combined with saturation mutations,and 6 libraries of saturation mutations was constructed reasonably.The HheC activity screening method established in this thesis was used to screen the dominant mutants with efficient ring opening of epoxides.In this study,1,2-epoxybutane was selected as the model substrate to screen the six libraries.Cause wild-type HheC shows mediocre catalytic activity for this substrate and low enantioselectivity.About 1400 clones were screened in every library,and a total of about 8400 clones were screened to obtain 8 advantageous mutants with efficient ring opening activity.Among them,the triple mutant R107K/D182N/E197K obtained from the error-prone PCR library even increased the activity of the substrate ECH by 4.1times,and the ring-opening activity of the substrate ESO was also increased by 2 times.In addition,mutants T134C/N176Q,N176Q,P184K,and F186Y have increased the activity of the substrate ECH by more than 3 times.Then,through homology modeling and molecular docking,the molecular mechanism of the increased catalytic activity of the mutants was initially explored.It was found that the interaction between the amino acids near the active site of the mutants and the substrate was enhanced,which should be the main reason for the increased enzyme activity.Finally,molecular dynamics simulation was used to further explore the reasons for the increased enzyme activity of the mutant,and found that the mutation did not affect the overall stability of the protein but made the mutant more stable than the wild type.According to the RMSF values of the two systems,it is found that the Asp182 which is far away from the active site in the thriple mutants is actively involved in the catalytic process,and the 121 and 123 sites that are not very active in the wild-type system are quite active in the mutant system.This may be the structural basis for the improved catalytic performance of the mutant,which provides a theoretical basis for the in-depth research and further transformation of HheC.