Key Amino Acids Affecting Regional Selectivity And Potential Cyclocylation Of ?-Farnesene Synthase

As the largest group of naturally occurring compounds,sesquiterpenes are widely used in food,agricultural medicine,cosmetics and other fields because of their physiological activities such as anti-tumor,antibacterial,herbicide,and anti-malaria.Sesquiterpene synthase directly regulates the synthesis and product specificity of sesquiterpenes.The analysis of the catalytic mechanism and potential cycalization mechanism of sesquiterpene synthase can guide the rational design of sesquiterpene synthase in practice and elucidate the root causes of the diversity of sesquiterpenes in theory.A small amount of amino acid mutation around the active center of sesquiterpene synthase can change the catalytic mechanism of the enzyme and form an enzyme with new catalytic function.Site-directed mutagenesis is currently commonly used to identify amino acids that affect the catalytic mechanism and cyclocylation capacity of sesquiterpene synthases.The conserved motifs of sesquiterpene synthase,such as RXR and DDXXD,plastic amino acids forming active center pocket,are often used as the first choice for site-directed mutagenesis experiments.The analysis of the three-dimensional structure of sesquiterpenoid synthase can provide a basis for the study of the relationship between structure and function of sesquiterpenoid synthase and its catalytic mechanism.It has been reported that Artemisia annua(E)-?-Farnesene synthase(Aa BFS)catalyzed the substrate(E,E)-FPP as a linear(E)-?-Farnesene,and Y402 L mutant catalyzed the substrate(E,E)-FPP to form a variety of cyclated products,such as zingerene,bisabololene,etc.However,T296 V mutation could make the cyclization ability of amorphodiene synthase disappear,and the catalytic product changed from cyclization product to linear product(E)-?-Farnesene.This suggests that the cyclization mechanism of sesquiterpene synthase is determined by key amino acids.In 2004,it was reported that Malus domestica ?-Farnesene synthase(MdAFS)catalysis substrate(E,E)-FPP was found in apple tissue.?-Farnesene and(E)-?-Farnesene were both linear in structure,but the double bond position was different.Can the regional selectivity of MdAFS be altered by site-specific mutation? Do MdAFS also have the potential to be cycled through mutations at key sites?With these questions in mind,two kinds of ?-Farnesene synthases MdAFS and Cucumis melo ?-Farnesene synthase(Cm AFS),which have different sources but consistent catalytic substrates(E,E)-FPP function,were studied in this study.The plasticiable amino acids around the space and conserved motif of MdAFS and Cm AFS were selected for point mutation.The mutants were expressed and purified by E coli.The proteins were catalyzed in vitro,and the specificity of the catalytic products was analyzed by GC-FID and GC-MS.A new expression vector was constructed to select the crystallization conditions of the wild-type recombinant proteins pET15b-MdAFS and pET15b-Cm AFS,hoping to analyze their three-dimensional structures and determine the specific action mechanism of amino acids that affect the enzymatic catalysis mechanism.The results showed that the mutants MdAFS-C298 W,MdAFS-S428 L,MdAFS-S428 F and MdAFS-Y329 R catalyzed substrates(E,E)-FPP to selectivity form(E)-?-Farnesene of the enzyme.MdAFS-C298W+L433A and MdAFS-S428L+C298W catalyzed the same substrate,and the relative proportion of(E)-?-Farnesene in the product increased significantly,indicating that the double mutation would further increase the content of(E)-?-Farnesene in the product.Mutants Cm AFS-F279 W and Cm AFS-F411 G catalyze the formation of terpenes from substrates(E,E)-FPP also selectively form(E)-?-Farnesene.When the intermediates trans-(S,E)-NPP were catalyzed,the content of the cyclated product bisabolol of the mutants MdAFSC298 W and MdAFS-S428 L increased significantly compared with the wild type.Cm AFSF279 W and Cm AFS-F411 amino acid mutants catalyzed the production of bisabolol,which was not found in the wild type.MdAFS protein was purified,and no crystal growth was found after screening under crystallization conditions.Cm AFS protein was purified with crystal growth.The above results indicate that MdAFS C298,S428 amino acids;The F279 and F411 amino acids of Cm AFS are the key amino acids that affect the catalytic region selectivity and potential cycalization ability of the enzyme.The catalytic mechanism of the same sesquiterpenoid synthases is similar.