Asymmetric Epoxidation Of Anethole Derivatives Catalyzed By Engineering P450 BM3 Enzymes

Objective:Anethole occurs naturally allylbenzene derivatives as a major component of essential oils from fennel and staranise.The epoxidation products of anethole can be used as precursors for synthesis of some drugs and active substances.Cytochrome P450 BM3 monooxygenase from Bacillus Megaterium is a kind of natural fusion protein consisting of a P450 heme monooxygenase and a NADPH-dependent diflavin reductase.It catalyses oxidation of different substrates,such as the hydroxylation of long chain fatty acid and the epoxidation of olefin.There is no report for the study on the asymmetric epoxidation of anethole analogues by P450 BM3.In this study,the P450 BM3 and its mutants were used for the epoxidation of anethole and its analogues.Methods:The F87V,F87A,F87G,F87M and F87A/T268M mutants of P450 BM3 were obtained by site-directed mutagenesis and the proteins were purified by IMAC.In addition,the kinetic constants for the asymmetric epoxidation of cis-?-methylstyrene catalyzed by the wild type and the mutants of P450 BM3 were calculated from the resulting regression of the Michaelis–Menten equation.The product ee was determined on chiral HPLC and the yeilds of products were analyzed by GC-MS.Results:DNA sequencing results showed that the F87V,F87A,F87G,F87M and F87A/T268M mutants of P450 BM3 were successfully constructed.The concentration of enzyme was 200-250?M.Comparing the ability of the asymmetric epoxidation of the mutants catalyzing cis-anethole analogues.The kinetic parameters of P450 BM3 parent was K_m=4.11 mM,K_cat=4.25 min^-1.The kinetic parameters of the F87V mutant was K_m=3.13 mM,K_cat=5.11min^-1.The kinetic parameters of the F87A mutant was K_m=6.81 mM,K_cat=1.07 min^-1.The kinetic parameters of F87G mutant was K_m=1.89 mM,K_cat=5.45 min^-1.The enantioselectivity and conversion were measured by HPLC and GC-MS,the mutant F87G was found to yielded relatively higher catalytic activity and ability of cis-anethole analogues and obtained 1R,2S configuration.The ee of cis-4-chloro-?-methylstyrene,cis-4-bromo-?-methylstyrene and cis-?-methylstyrenes catalyzed by the F87G mutant were more than 90%.Generally,the epoxidations of the styrenes analogues catalyzed by the F87A mutant give much higher enantioselectivity?72–84%ee?than those of the cis-?-methylstyrene analogues?10.77-61.97%?.Interestingly,the F87G mutant catalyze the cis-?-methylstyrenes?45.60-83.62%ee?and 1S,2R configuration.Conclusion:The replacement of Phe87by other small hydrophobic amino acids was conducive to the improvement of enantioselectivity and conversion on asymmetric epoxidation of cis-anethole analogues.At the same time,both the size of the amino acid at position 87 and the size of the substituents on the benzene ring of the substrate have an effect on the inversion of the epoxide configuration.The results of this study is helpful for us further study the catalytic mechanism of P450 BM3 and the relationship between the substrate and the structure of the enzyme.