Studies On The Crystal Structure And Molecular Mechanism Of Engineered Enzymes

Various catalytic properties of enzymes can be efficiently improved by directed evolution.It draws great attention,especially after the award of the Nobel Prize in Chemistry in 2018 to Professor Arnold from California Institute of Technology.However,it is still a question why the engineered enzymes have better properties,and how the mutations change the protein structure.To get clearer answers,researches of crystallography or computational chemistry are necessary.By analyzing the crystal structure of the mutants,it can be better understood about the effect of mutations on the shape,charge and overall conformation of mutants.The combination of directed evolution and crystal structure analysis provides a better molecular basis of enzyme engineering.Three CALB(Candida antarctica lipase B)expression systems were constructed in this work.A co-expression system of pETM-11-calb and pGro7 in E.coli.BL21(DE3)was finally chosen,considering the aspects of expression level,culture cycle,purification process and the convinence of directed evolution.Following the work above,the alcohol binding pocket of CALB was designed,and a S-selective mutant,opposite to the wild type,was achieved for the kinetic resolution of 1-phenylethanol acetate.The crystals of four CALB mutants were obtained,which show highly distinct stereoselectivity for the ester products with two stereo-centers.Four apo-protein structures and one complex structure with the product analog were analyzed by X-ray diffraction.The binding ability of each mutant to the product with dominant conformation was solved by computational chemistry methods.The binding modes in the catalytic pockets were analyzed,and the contribution of each mutation to the specific stereoselectivity was elucidated.Two kinds of CALB mutants,QW10 and QW4,which can efficiently catalyze the hydrolysis of benzoate,were successfully crystallized and the crystal structures were sovled by X-ray diffraction.The structure was analyzed and it was found that the difficulty for substrate entering the catalytic cavity was consistent with their catalytic activities.Further,the results of MD simulation proved that each mutation site contributes to the substrate entering the catalytic position from steric hindrance,hydrogen bonding,hydrophobic interaction and the size of entrance,improving the catalytic activity of the reaction.The formation of hydrolysis intermediate state in QW4 and QW10 was studied with QM/MM.It was found that a two-step reaction mechanism,deprotonation followed by nucleophilic attack,happened in QW4,while a concerted mechanism was found in QW10.Genetic engineering methods were used to improve the crystallinity of CHMO(Cyclohexanone Monooxygenase),by surface entropy reduction mutation,truncation mutation and fusion expression.After making great effort,the protein crystal with diffraction ability was finally obtained.However,the crystal structure was not able to be solved because of a low resolution.In this paper,eight protein crystal structures,one of which was complex structure,were obtained.All the structures have been deposited to the PDB database.