Molecular Modification Of Halohydrin Dehalogenase And Its Application In Asymmetric Biosynthesis Of Chiral Epichlorohydrin

Chiral epichlorohydrin?ECH?is a chiral intermediate in the synthesis of chiral pharmaceuticals and fine chemicals and advanced materials.Halohydrin dehalogenases?HHDHs,EC 4.5.1.X?are reported to catalyze the transformation of1,3-dichloro-2-propanol?1,3-DCP?to chiral ECH.However,the low stability and low optical purity of chiral ECH caused by the reverse reaction limited the application of HHDHs.In the present study,the molecular modification of HHDH and the biosynthesis of optically pure?S?-ECH were performed:After analyzing the key amino acid sites of halide ion channels and substrate conformation,site-directed saturation mutation and iterative saturation mutation were carried out using HheC?P175S/W249P?(HheC_PS)as the template.Mutants HheC_PS I81W,HheC_PS E85P,HheC_PS F86N and HheC_PS V94R with high-stereoselectivity were obtained.Compared with HheC_PS,the relative activity of mutants were 34.65%,69.15%,39.78%and 25.43%,respectively,in the process of the biosynthesis of optically pure?S?-ECH.The HheC_PS mutants were recombinant expressed,isolated,purified and the kinetic parameters were determined.The Michaelis constants(K_m?1,3-DCP?)of HheC_PSS I81W,HheC_PS E85P,HheC_PS F86N,and HheC_PS V94R for forward reactions were reduced from 20.83 mM to 19.35 mM,20.72 mM,16.87 mM and 15.84 mM,respectively.The catalytic efficiency(k_cat?1,3-DCP?/K_m?1,3-DCP?)of HheC_PS I81W,HheC_PS E85P,HheC_PS F86N,and HheC_PS V94R were 0.73 mM^-1s^-1,0.71 mM^-1s^-1,0.64 mM^-1s^-1 and 0.32 mM^-1s^-1,which were 0.88-,0.85-,0.77-and 0.36-fold lower than HheC_PS,respectively.The Michaelis constants(K_m??S?-ECH?)of the reverse reactions of HheC_PS I81W,HheC_PS E85P,HheC_PS F86N and HheC_PS V94R were improved from 1.38 mM to 4.01 mM,6.14 mM,7.31 mM and 6.75 mM,respectively,which were 2.91-,4.45-,5.30-and 4.89-fold higher than HheC_PS,respectively.The catalytic efficiency(k_cat??S?-ECH?/K_m??S?-ECH?)were 0.05 mM^-1s^-1,0.03mM^-1s^-1,0.03 mM^-1s^-1 and 0.03 mM^-1s^-1,which were 0.38-,0.23-,0.23-and0.23-fold lower than HheC_PS,respectively.Therefore,the reverse reactions of the mutants were retarded greater than the forward reaction,the optical purity of?S?-ECH was significantly improved.The mutans,HheC_PS I81W,HheC_PS E85P,HheC_PS F86N and HheC_PS V94R were applied for the preparion of high optically pure?S?-ECH.At 37^oC and 20 mM1,3-DCP,?S?-ECH was obtained with the enantiomeric excess?e.e.?>99.99%,firstly,and the yields were 63.45%,69.15%,67.58%and 57.50%,respectively.The stereoselective catalytic mechanism of HheC_PS mutants were investigated using homology modeling and molecular docking technologies.The 81^st,86^th and94^th amino acid residues were predicted to affect the formation of the halide ion channels,and the 85^th amino acid residue was considered to alter the conformation of?S?-ECH in the substrate pocket.As a result,the modification on the above amino acid residues regulated the kinetic parameters of the forward and reverse reacitons,and the stereoselectivity of HheC_PS mutants were improved.To our knowledge,it was the first time to prepare optically pure?S?-ECH using biosynthetic technology catalyzed by HHDH.