Modification Of Halogenol Dehalogenase For The Synthesis Of Chiral Building Blocks Of Oxazolidinone

Halohydrol dehalogenases have good catalytic properties for both dehalogenation and ring opening reactions catalyzed by nucleophilic reagents.When the nucleophilic reagent is OCN~-,it can catalyze the epoxide to produce the parent ring of oxazolidinone.Oxazolidinones are a new class of antibacterial drugs with the efficacy of inhibiting multi-drug resistant Gram-positive bacteria.They have received extensive attention and research in the pharmaceutical industry at home and abroad.At present,oxazolidinones mainly rely on chemical total synthesis,and the related methods are not only cumbersome and inefficient,but also use some toxic reagents in the synthesis process of some methods.Therefore an efficient and green synthesis method has been pursued.The biocatalytic reactions of halohydrol dehalogenases have laid a solid foundation for the realization of the biologic synthesis of the parent ring of oxazolidinones.However,most of this research has stayed in the catalytic reactions of wild ecological enzymes and the wild Hhe C shows low activity and enantioselectivity toward a series of epoxides.Thus,a modification study aiming to improve the catalytic properties of halohydrol dehalogenase toward the model reaction of OCN~-mediated ring opening of epoxides is necessary.For this,we constructed high-quality mutant librariesby by means of both semi-rational design and directed evolution.and the related high-throughput screening methods was optimized.Firstly,based on the information of the crystal structure(PDB ID:1pwz)of Hhe C co-crystallized with R-styrene oxide and the docking results of Hhe C and OCN~-molecule,the amino acids around the substrate binding pocket were applied as the key amino acids for the modification using CASTing strategy,aiming to investigate the effect of these residues on the enzyme catalytic properties toward the above model reaction.In addition,the mutant library constructed by error-prone PCR was proposed to further mine key amino acids far away from the active center.These strategies will provide a high efficiency for the acquisition of beneficial mutants.Secondly,we selected 1,2-epoxybutane as the model substrate to screen the nine semi-rational design libraries and one random mutagenesis library constructed by error-prone PCR.The obtained dominant mutants were analyzed for enzyme activity,enantioselectivity,enzyme kinetic parameters,and substrate range.Among them,R107K,obtained from the error-prone PCR library,showed a nearly fivefold increase in catalytic activity for the production of oxazolidinones from the substrate 1,2-EPH,and mutants N176Q,P184K,F186Y,T134A/P135F showed more than twofold increase in catalytic activity.The catalytic activity of F12L toward 1,2-epoxy-octane and epichlorohydrin was also increased by about two-fold compared with the wild type enzyme.Finally,through homology modeling and molecular docking,we analyzed the effects of mutations on enzyme catalytic properties.We found that the mutants T134V and F12L,both have closer distance between the substrate and the catalytic triplet,and the size of the substrate binding pocket has changed big,which is more favorable for the entrance of the substrate,These changes might contribute to their increased enzyme activity.These studies provide a theoretical basis for further modification of HheC.