Halogen Alcohol Dehalogenation Enzyme Hhea Biological Catalytic Properties And Transformation Of The Research

Halohydrin dehalogenerase has been reported as a valuable tool for bioremediationand biocatalysis. It catalyzes the dehalogeneration of vicinal halogen alcohol and thenucleophilic displacement of epoxides at Cβ atoms. Although HheC which had beenmodified for industrial process is highly selective to R-type substrates, there is noexcellent variant that has high S-type substrates selectivity having been obtained. Theabsence of these variants limited the further industrial application of Hhes in organicasymmetric synthesis. The wild-type HheA had been supposed as a good template forevolution of S-type substrate selectivity. HheA and HheC have very similar X-raystructures, contrasting to the remarkable difference in their amino acid sequences.Previous reports revealed that three loop regions constructs the active-site of HheCexist in HheA also. Here a structure-guided “semi-rational” evolution focusing onthese loops was described. The mutagenesis libraries of HheA were constructed using“small-intelligent” pathway to enhance the efficience of library screening, which isnecessary during the evolution. This pathway eliminated the redundancy and stopcodons to enhance the quality of library by intelligent designing of primers. As theresults of evolution, we got several positive variants which had both highenantioselectivity and activity. The best variant HheAN178A had a5-fold higheractivity and100-fold higher S-type enantioselectivity (Ecal.from2to＞200) thanHheA in dehalogenation. Also, this variant had a50-fold higher S-typeenantioselectivity than HheA in nucleophilic displacement of epoxides. Intriguingly,this variant had a high enantiomer excess (e.e.≥93%) in both reactions. The moleculardocking analysis revealed the molecular mechanism of the high enantioselectivity andactivity of these variants. In additional, these analysis helped us to identify more keyresidues those determine the enantioselectivity and activity of HheA. We believe thatthese works could provide important information to further engineering of HheA.