Semi-rational And Rational Design Of Enzyme Activity And Enantioselectivity

The optical purity of chiral compounds is a crucial criterion in industries of pharmaceuticals, fine chemicals and materials due to the significant distinctions in the chemical properties and biological activities of the enantiomers. Enzyme-mediated production of chiral compounds is a favored method over chemical synthesis because of the high specificity and mild reaction conditions. Highly active and enantioselective enzymes are critical in the production of chiral compounds so that it is necessary to optimize these properties of enzymes. Protein engineering manipulating the interactions between enzymes and substrates at molecular level could optimize the catalytic properties of enzymes. In this research, the activity and enantioselectivity of esterases and dehydrogenases, which are widely applied in chiral compounds production, were designed and optimized by semi-rational and rational strategies.The first part of this research was the compensation of the activity and enantioselectivity trade-off observed in the previous study on directed evolution of the enantioselectivity of esterase RspE. A trade-off between the activity and enantioselectivity was encountered by mutant YH (E 8.7, specific activity 142 μmol min-1 mg-1) and mutant CVH (E 30.8, specific activity 24 μmol min-1 mg-1). After structural analysis, site-directed mutagenesis was conducted on hot-spots and screening of the mutants libraries resulted in a mutant HMV (E 36.8, specific activity 113 μmol min-1 mg-1) with high activity and enantioselectivity.Kinetic analysis revealed that the reason for the activity and enantioselectivity trade-off was the different strategies adopted in the improvement of enantioselectivity. Consequently, the molecular dynamics simulations and analysis of conformations and energies were conducted and the investigations on the mutated sites revealed these residues optimized the complex between HMVY and the preferred substrate by strengthening their interactions and relocating the binding site.As the second part of this research, enantioselectivity of esterase BioH in the asymmetric hydrolysis of prochiral dimethyl 3-phenylglutarate was redesigned by rational strategies. The structural analysis of protein-ligand complex revealed that the orientation of the substrate substituted group in the protein activity pocket determined the enzyme enantioselectivity. Aromatic residues were introduced to specifically interact with the phenyl ring in the pro-S substrate by π-π stacking, resulting in a mutant L86F with significantly enhanced enantioselectivity. The enantiomeric excess of the chiral product was improved from 25%(WT) to 93% (L86F).To further testify the efficiency and generality of the described rational strategy, the enantioselectivity of esterase RspE was also designed by tuning the interactions between the protein and the substitution group of dimethyl 3-phenylglutarate. The enantiomeric excess of the chiral product was improved from 13% (S) by the WT to 99% (S) by Y27R and reversed to 50% (R) by M121F. These results confirmed the efficiency and generality of this rational design strategy.Moreover, the activity of D-mandelate dehydrogenase in the oxidation of R-o-Cl-mandelic acid was improved by rational design. Halogen bond was introduced between the protein and the halogenated substrate by the substitution of alanine by histidine. Mutant A89H was obtained with 5 times higher activity compared to the wild type.Activity and enantioselectivity of esterase BioH, esterase RspE and D-mandelate dehydrogenase were successfully redesigned by semi-rational and rational strategies. These improvements would benefit their application in the production of chiral compounds.