Improving Activity And Enantioselectivity Of Halohydrin Dehalogenase Hhec By Using Semi-rational Design

Halohydrin dehalogenerase has been reported as a valuable tool for bioremediation and biocatalysis. It catalyzes the dehalogeneration of vicinal halogen alcohol and the nucleophilic displacement of epoxides at Cβ atoms. Although Hhe C which had been modified for industrial process is highly selective to R-type substrates, there is no excellent variant that has high S-type substrates selectivity having been obtained. The absence of these variants limited the further industrial application of Hhes in organicasymmetric synthesis.Haloalcohol dehalogenase Hhe C from Agrobacterium radiobacter AD1(Hhe C) shows a great potential in the production of valuable compounds with R enantiopreference. To gain a deep understanding on how the enzyme enantioselectivity is controlled, ten noncatalytic active-site residues located within 5 ? of the binding substrate in the crystal structure of Hhe C(PDB code: 1PWZ) were subjected to two rounds evolution. In the first round, 5 CASTing libraries were constructed and screened, yielding 42 positive variants for their abilities to efficiently catalyze the dehalogenation of 1,3-dichloro-2-propanol(1,3-DCP). In the second round, the enantiopreference of Hhe C was further explored by constructing 4 combinatorial ISM libraries based on the enantioselectivity of 14 out of 42 positive variants toward 2-chloro-1-phenylethanol. After screening about 800 colonies using both enantiomers of 2-chloro-1-phenylethanol, three outstanding mutants(T134V/L142M; L142F/N176H; 84V/86 P /134A/176A) with diverged enantioselectivity were obtained. The first two double mutants displayed an about 2-fold enantioselectivity improvement toward 2-chloro-1-phenylethanol without significant loss of enzyme activity as compared with the wild-type enzyme. Strikingly, the 84V/86P/134A/176 A mutant showed an inverted enantioselectivity(ER = 65(WT) to ES = 101) and an about 70-fold enhanced catalytic efficiency toward(S)-2-chloro-1-phenylethanol. Molecular docking analysis revealed that those mutations produced some interference for the binding of non-favored enantiomers which could explain the observed enantiopreference. Our work demonstrated that the enantiopreference of Hhe C could be modulated by manipulating the side chain properties of active-site residues.