Exploring The Interaction Of Four Loops In Halohydrin Dehalogenase By Iterative Saturation Strategy

Halohydrin dehalogenases(HHDHs)efficiently catalyze the dehalogenation of vicinal halo alcohols to epoxide by the intramolecular substitution mechanism.Also,HheC can catalyze epoxide ring-opening reaction with a series of anion nucleophiles.Therefore,the enzyme plays an irreplaceable role in the preparation of chiral pharmaceutical intermediates such as optically pure ?-substituted alcohols.It also has applications in the degradation of polluting organic halides.The crystal structure of HheC lays the foundation for studying the catalytic mechanism.The homotetramer enzyme HheC performs catalytic function relying on the Ser132/Tyr145/Arg149 catalytic triad.In addition,four loops around the catalytic center of the enzyme have different degrees of influence on the catalytic function.Loop1 and loop2 regions are close to the hydrogen bond network of the substrate-binding pocket.The loop3 region is the halide ion binding area,which is closely related to the rate-limiting step of the enzyme catalysis process;the loop4 region,a unique amino acid sequence of HheC,is closely related to the composition of the substrate release channel.It has a significant effect on enzyme stability.According to existing research,four loops are selected as research points.This thesis combined with experimental method and computational biology selected 8 key sites,and constructed 12 ISM libraries.After screening nearly 5300 samples with1,3-DCP as a model substrate,8 brilliant variants were obtained,which the best one12-3(P84A-F86A-T134L-W139V)displaying approximately 8.4-fold higher activity than the wild-type enzyme.The results showed that the library formed by any two loops except loop3 yielded dominant mutation.Therefore,it is speculated that loop1,loop2,and loop4 have interaction during catalysis.To further explore the structural relationship between the loops,the thesis constructed the crystal structure of the mutation,simulated molecular docking,and predicted the internal tunnel of the enzyme via computer software.The analysis results showed that the amino acid changed the hydrogen bonding network between loops.Also,the absence of hydrogen bonds made the substrate pocket more flexible and the hydrophobic range expands,the P84,F86,W139,and W249 sites all prefer small side-chain amino acids.After site overlapping,the steric hindrance of the enzyme-substrate pocket is reduced,which is conducive to the release of halide ions.In addition,the catalytic ability of the enzyme is significantly enhanced.The predicted results of the CAVAR internal tunnel of the enzyme indicated that the mutated of 139 changes the orientation of tunnel-004.The tunnel no longer turns around at the site but goes straight into the solvent environment.Put the exit turns to the loop4 region simultaneously to obtain a new export with a loose structure and great flexibility.At the same time,the increased tunnel space is more conducive to the entry and exit of halide ions into the enzyme molecule,thereby improving the catalytic efficiency.Similar phenomena appeared in the structures of 14-1 and 41-1.These are sufficient to prove that the amino acids in the loop1,loop2,and loop4 regions interact in the catalytic process of HheC.