The Role Of Thr215 And Phe153 Site In CYP 119 In Epoxidation Of Styrenes

Objective: Aromatic epoxides are important intermediates for preparing various drugs,spices and etc.which can be synthetized via epoxidation of olefins with catatyst of CYP119 enzyme.The effect of two consecutive conserved threonine residues on the CYP119 enzyme activity center was studied in detail,and no further study was conducted on the other conserved Thr215.The crystal structure of CYP119 shows that Phe153 is in the channel of the substrate,so can the amino acid mutation of the site change the catalytic efficiency of the enzyme? In this study,a series of mutants at position 215 and 153 were constructed to clarify the role of conserved Thr215 and Phe153 in the CYP119 enzyme.Methods:Site directed mutagenesis was used to obtain T215 G,T215V,F153 A,F153V,T213M/F153 A.The thermal stability of the mutants were investigated,meanwhile,the conversion rate and enantioselective of epoxidation of terminal aromatic alkenes and non-terminal aromatic alkenes were tested.The catalytic ability of 153 mutants to styrene and cis-?-methylstyrene was studied by molecular dynamics and molecular docking.The theoretical results were verified by in vitro enzymatic reaction.Results: The results showed that the T215 V and T215 G mutants are similar with the wild type in catalyzing the terminal aromatic alkenes.However,there is difference among the conversion rate and enantioselective of the mutants T215 V,T215G and wild type CYP119 catalyzing the non terminal aromatic alkenes.The T215 G mutant is discovered to show highly conversion rate epoxidation of styrenes with up to 99.6% and enhanced the conversion rate and enantioselective for epoxidation of cis-?-methylstyrene.The mutant T215 V is found to reduce the enantioselectivity of cis-?-methylstyrenes.The conversion and enantioselectivity of the mutant F153 A and F153 V catalyzed styrene were similar to those of the wild type,mutant T215 G and T215 V.The conversion and enantioselectivity of the mutant T213M/F153 A catalyzed styrene were lower than that of the wild type and its single mutant.However,for cis-?-methylstyrene,the conversion and enantioselectivity of the mutant F153 A,F153V were higher than that of the wild type,slightly lower than the mutant T215 G,While the conversion of the double mutant T213M/F153 A decreased to 13.61% and the enantioselectivity was as high as 92.4%.Molecular docking data shows that the mutant F153 A catalyzes the highest efficiency of substrate styrene and cis-?-methylstyrene,and F153 V has also improved the catalytic efficiency of parental enzymes,and the F153 V was higher than that of the wild type,while the mutant T213M/F153 A were decreased significantly.Conclusion: The hydrogen bonding interactions and the chain size of the Thr215 site have certain influence on the epoxidation activity of special substrates.The size of the 153 side chain phenylalanine has an important effect on the substrate access channel.