Exploring The Function Of Amino Asids In The Halide Binging Site Of Haloalcohol Dehalogenase HheC

Halohydrin dehalogenases(HHDHs)are lyases that catalyze the cleavage of carbon–halogen bond of halohydrins.They can also catalyze the reverse reaction in the presence of nucleophiles such as cyanide,azide,and nitrite ions.HHDHs have been recognized as ideal tools for the degradation of various halogenated environmental pollutants.Moreover,they can be used as biocatalysts for the kinetic resolution of halohydrins and epoxides,and for the preparation of various ?-substituted alcohols.The active center of the halohydrin dehalogenase HheC consists of four flexible loop regions,in which the loop3 region formed by 175-188 sites is the halide ion binding region.Kinetic studies have shown that the release of halide ions is the rate-limiting step of the enzymatic reaction and the release of halide ions requires conformational changes of the active site,indicating that the flexibility of the P175-P188 carbon skeleton is crucial to the enzyme activity.Halohydrin dehalogenase(HHDH)can degrade halohydrins to their corresponding epoxides and catalytic reverse reaction in the presence of affinity reagents.Loop 3 plays an extremely important role in these reactions.Howerer,so far this area has not been well studied.Therefore,in order to explore the function of amino acid at the halide ion binding site,a systematic study has been conducted in this work.Firstly,the effect of amino acids of loop3 on both activity and enantioselectivity were analyzed by alanine scanning.1,3-DCP and rac-2-CPE were used as model substrates.The results showed that the mutations on positions179,181,182,and 184 have no significant effect on the catalytic activity and enantioselectivity compared to the wild type HheC,indicating thatthese sites might not be crucial for enzyme catalysis.The kinetic resolution of N176 A showed that the enantioselectivity of the mutant was reversed compared to the wild-type enzyme(from R preference to S preference),which could be the hot spot for modulating the stereoselectivity of HheC.The P175 A mutant showed substrate preference.For the rest of other mutants,they were all inactive,indicating an important role of the residue on these positions for enzyme catalysis.Thus,these key residueswere further studied.In order to further explore the function of key sites in loop3,these residues were subjected to saturation mutagenesis.The resulting positive mutants were tested withaspects of both,activity and enantioselectivity.The results showed that the residues on positions 176,185,186,187 and 12 sites might be important in regulating the enantioselectivity of Hhe C.After screening 200 variants from libraries on positions 180,183 and 188,no active mutants were obtained.SDS-PAGE results showed that these inactive mutants displayed a similar expression level as the wild-type enzyme.Further native-PAGE and gel filtration analysis proved that the inactivation of the variants is more likely to be caused by tetrameric dissociation of HheC since the enzyme is only active as tetramer.Thus,the residues on positions 180,183 and 188 might play a crucial role in maintaining the structure of the enzyme.The results of mutants on positions 175 and 176 showed that the residues on these two positions are not only crucial for enzyme activity but also for substrate selectivity.Interestingly,only the replacement with either F or Y on positions 12,177,185,186,187 and 12 was obtained,and all these mutations displayed an improved effect on the enzyme activity of HheC,suggesting the importance of such a hydrophobic microenvirment on the enzyme catalysis.