Exploring The Cooperated Conformational Change Of Four Loops In The Catalytic Process Of Haloalcohol Dehalogenase HheC

Halohydrin dehalogenase?HHDHs?catalyzes the hydrolysis of halohydrins into epoxides through the molecular nucleophilic substitution mechanism,and its reverse reaction by accepting different nucleophiles,such as CN^-,N₃^-and NO₂^-.So it is of great value in the treatment of organic halogenated pollutants and the synthesis of chiral drugs intermediates.The X-ray structure of HheC showed that the active site of the enzyme is embedded inside of the enzyme molecule,which is surrounded by four Loop fragments,forming substrate binding pocket and halogen ion binding site.Previous studies showed that 1.Release of halogen ions is the rate-limiting step in catalytic dehalogenation;2.Loop3 fragments is the binding site of halogen ions,which plays a key role in the binding and release of halogen ions during the catalytic process,and the conformation changed during the catalytic process;3.Loop4 was also proved to have significant regulatory effects on catalytic activity and thermal stability.At present,the Loop1 and Loop2 regions were not explored in depth and the structural basis of the conformational changes of the whole active site was not elaborated.In this paper,the conformational change and the structure dynamics of the channels of halogen ions in the process of enzyme catalysis were studied by combining experimental means and computer technology,the key problems mentioned above are discussed from the protein flexibility change.The key residues D80,I81,F82,P84,F86,T134,W139 and L142 that have significant influence on Loop1 and Loop2 were obtained through alanine scanning,and D80A,I81A and F82A mutants showed no significant activity;the activity of the other five sites were significantly increased by more than 2 times after mutation to alanine.Which were constructed the saturated mutant library and conducted activity screening.The results showed that no significant active mutants were detected at D80,I81 and F82,which indicates the importance of these three sites in catalyzing of HheC,and the mutants with different degree of activity were also screened in the saturated mutant library of the other five sites.In order to further explore the effect of conformational changes on enzyme activity,the dominant mutants located on the same loop were combined and a series of dominant mutants with increased enzyme activity by 6-15 times were obtained.The three-dimensional structure of dominant mutants were optimized by molecular dynamics?MD?simulation and the interaction between amino acids was analyzed.Results showed that the flexible increase of the active center of the mutants P84A,F86A,T134A,W139A and L142A,and a series of dominant mutants of W139.We speculate that the conformational change of the protein by reducing the side chain of the residue lead to the enlarge of catalytic space,or the destroy of the interaction with surrounding amino acids.The inference was proved by the increased activity of multiple mutants such as P84A-F86A.in conclusion,the flexible change of the substrate binding pocket or halogen ion binding site is the main reason for the increase of enzyme activity.By analyzing the trajectory of Br^-released from the active center into the solvent through molecular dynamics simulation,the positions of Br^-at different times were obtained and the release channels of halogen ions were predicted by surrounding amino acids of Br^-.The results showed that F82-P84 on Loop1,T131-T134 and W139 on Loop2,P175-L178 and Y185-Y187 on Loop3,W249 on Loop4 and F12 residue could played a key role in halide releasing.The comparison of three-dimensional structures also showed that the conformation of these sites changed significantly before and after the release of halogen ions.It is concluded that these amino acids may be involved in the conformational changes associated with the release of halogen ions,and related studies can lay a theoretical foundation for the subsequent transformation of halohydrin dehalogenase.