Directed Mutation And Mechanism Of Perakine Reductase For Carbonyl/Alkene Bond Reduction Selection

Selective asymmetric reduction of the carbonyl or alkene bonds of?,?-unsaturated carbonyl compounds can lead to chiral enols or saturated carbonyl compounds,which have important applications in the fields of drug synthesis and structural modification of natural products.However,since the bond energies of carbonyl and alkene bonds are relatively close,it is difficult to control the regioselectivity and stereoselectivity of ?,?-unsaturated carbonyl reduction reactions using chemical methods.Currently,transition metals and their organic complexes are mainly used as catalysts,but transition metals are expensive and easily contaminated,and their complex synthesis steps are complicated,making industrial applications difficult.Compared with chemical methods,enzyme-catalyzed carbonyl/alkene bond reduction has no regioselectivity problem,and generally has the advantages of strong stereoselectivity,mild reaction conditions,single and easy purification of products,high efficiency and environmental protection,etc.,which is more promising for industrial development.Perakine Reductase(PR)is derived from the medicinal plant Rauvolfia serpentina and is a member of the aldehyde and ketone reductase(AKR)superfamily,which uses NADPH as a cofactor to catalyse the reduction of various structural types of carbonyl Vcompounds.It has a broad substrate spectrum,high stereoselectivity,good stability,high heterologous expression,easy purification and preparation,and has a good basis for industrialization,but at present its catalytic efficiency cannot reach the level of industrial application,and still needs to be screened for efficient carbonyl reduction mutants.Based on the property that mutations in the catalytic centres of the AKR superfamily of carbonyl reductases and alkene bond reductases can convert the two activities,it is expected that targeted mutation of PR to alkene bond reduction activity will enhance the application of PR and provide a good performance biocatalyst for alkene bond reduction.In this thesis,based on the crystal 3D structure of the Perakine reductase complex,amino acid sequence comparison and computer molecular simulations were used to identify histidine at position 126 and arginine at position 127 as the key sites for substrate carbonyl binding of PR,and the above two sites were mutated by saturation mutagenesis,and benzylidene acetone was used as the substrate,and the product yield and substrate were detected by high performance liquid chromatography The relative activity of the mutant carbonyl or alkene bond reduction was determined by high performance liquid chromatography.The sequencing was validated and 19 mutants each at positions 126 and 127 were successfully obtained.Among the 126 mutants in PR,all but four were inactivated and all the other 15 mutants at position 126 were converted to alkene bond reduction activity,with the strongest alkene bond activity being H126G;the activity of the 127 mutants was more complicated,with nine mutants retaining carbonyl reduction activity,six mutants converted to alkene bond reduction activity,three mutants having both carbonyl and alkene bond reduction The carbonyl reduction activity of all 127 mutants was significantly lower compared to the wild type,with the highest carbonyl reduction activity,R127 L,having only 5.7 % of the relative carbonyl reduction activity of the wild type,and R127 M being the mutant with the strongest alkene bond reduction activity,with a relative activity ?40 % higher than that of the H126 G mutant.The catalytic kinetics of the PR mutant showed no significant difference in the affinity of the mutant and the wild type for the substrate.To further elucidate the mechanism by which amino acids at positions 126 and 127 alter the catalytic function of PR,we selected mutants with better alkene bond reduction activity at positions 126 and 127 for structural biology studies to investigate the conditions for crystallization of PR mutant-cofactor-substrate complexes,but so far we have been unable to obtain crystals of the complexes.In the absence of crystallization,we have adopted computerized molecular docking and kinetic simulation methods to investigate the catalytic mechanism of the PR mutant.The results show that the relative positions of the carbonyl and alkene bonds between the substrate molecule and the cofactor nicotinyl ring in the mutant determine the reduction selectivity.This thesis has successfully achieved a targeted mutation of the PR carbonyl/alkenyl bond reduction option to provide a novel biocatalyst with excellent catalytic performance for the selective reduction of ?,?-unsaturated carbonyl compounds,significantly enhancing the application of PR.