Structure-guided Engineering Of Halide Binding Site Of Halohydrin Dehalogenase HheC

Halohydrin dehalogenases(Hhes) are a family of enzymes originally from bacteria which are capable of converting environmental halogenated pollutants to industrial valuable epoxides. On the other hand,Hhes also catalyze the reverse reaction and yield corresponding chiral drug precursors. Active center of HheC contains four flexible loops and the Loop3(P175-P188) is the halide ion binding site. Kinetic studies have shown that the release of halogen ions is the rate-determining step of the enzymatic reaction. Hhes can accept a variety of nucleopilic reagents apart from a halogen atom, and its products could be used as intermediates to prepare for optically pure building blocks. Quantum chemical models have been elaborated that certain amino acid residues lead to important conformational changes during the reaction process. And the latest research shows that combination engineering of key amino acids can modulate enantioselectivity preference. But there is no syetematic study of halide ion binding site still now. Therefore, this program comprehensively investigates the role of halide binding site in HheC and mainly included the following work:Employing alanine scanning technology,we systematically explored the critical role of the amino acid halide ion binding region for activity and enantioselectivity, and revealed that P175 A, N176 A,S180A,S183 A,F186A and Y187 A remained good activity for aliphatic substrates 1,3-DCP while with a great loss of activity for aromatic substrate 2-CPE, which suggested that these residues may have an influence on the enzyme-substrate binding for different type of substrates. For L178, H179, E181, D182, P184 and Y185 sites, alanine-subsituded mutants had a good catalytic activity. Meanwhile, enantioselectivity of H179 A performed a certain improvement, but L178 A dramatically reduced its product and substrate stereoselectivity excess which was consistent with the result in theoretical calculation study. In addition, Y177 A and P188 A lost their activity for both substrates indicating that these two sites play a key role on catalytic function of Hhes.Based on the above information of essential residues, saturated libraries were constructed to further exanmine the function of residues located at position Y177, P188, F12, N176, L178, H179, Y185 and F186. Good variants F12 Y, L178 V, L178 I and F186 Y were obtained and their activity increased 1.4-3 fold compared with wild-type, and the reson for its increased activity was conformed by molecular docking. Besides, mutants with different types of substitution at N176 obtained by saturated mutagenesis exhibited manifest differences in enantioselectivity which suggested that N176 played a strong regulatory role on enantioselectivity. These results may lay an important foundation for further engineering study of Hhes.