Effects Of The Fourth Amino Acid Mutation In The Upper NSE/DTE Motif On The Enzymatic Properties Of The Sesquiterpene Synthase

Terpenoids are common secondary metabolites in plants and microorganisms.Which capable of catalyzing the substrate formation of structural and stereochemical diversity of isoprenoid natural products are called steroidal synthases.The study on the structure and function of sesquiterpene synthase can not only provide insight into the catalytic mechanism of terpene synthase,but also help to obtain a wide variety of terpenoids to benefit mankind.Glycine-439 and Leucine-515 are located on two different alpha helices on the surface of the active center cavity in Artemisia annua Amorpha-4 11-diene synthase,AaADS.Glycine-439 is also the position of the 4th amino acid residue upstream from the non-classical motif NSE/DTE.Our previous study found that the side chain of 439 amino acid residues in AaADS occupies a certain amount of space in the active center cavity.When the enzyme binds to the substrate(E,E)-FPP,it affects the catalytic specificity of the enzyme if the space increase to a certain extent,but it can be recovered by shifting the leucine-515 residue into a smaller side chain amino acid,which on the opposite alpha helix.This mutual synergy between amino acids can compensate for the negative effects of natural sesquiterpene synthase mutations.However,the crystal structure of AaADS has not been analyzed so far.The reason why the mutation of 439 amino acid residues affects the enzymatic properties of AaADS remains to be further studied.Two sesquiterpene synthases with known crystal structure were selected in this thesis: Artemisia annua Bisabolol synthase,Aa BOS and Nicotiana tabacum 5-epi-aristolochene synthase,NtEAS.After site-directed mutagenesis,Escherichia coli-induced expression,malachite green assay of the inorganic pyrophosphate content method for measuring the kinetic constants of the protease,and GC-FID analysis of the catalytic product,the following conclusions were drawn:1 Using(E,E)-FPP and(Z,E)-FPP as substrates,when the side chain of the fourth residue in the upstream of the AaBOS NSE/DTE motif invaded into a small spatial volume of the active center cavity,the activity of the enzyme and the specificity of the catalytic product are not affected.When the side chains increase to a certain extent,the catalytic activity of the enzyme decreases and the specificity of the catalytic product changes.The by-products increase(C439)and even enzymes lose their activity(V439,I439).After the mutant residue(C439,V439)was mutated to the smaller residue A of the 64 th residue of the NSE/DTE motif downstream of the NSE/DTE motif,the specificity of the catalytic product was nearly restored to the wild type,and the catalytic ability of the inactivated mutant was also restored(V439+A515).Although the catalytic activity has recovered,it is still weak overall.2 With(E,E)-FPP as the substrate,when the spatial volume of the fourth residue upstream of the NtEAS NSE/DTE motif invading the active center cavity changes specifically,the specificity of the catalytic product of the enzyme is greatly affected.After the side chain is increased to a certain extent,the catalytic activity of the enzyme is decreased(I440);after the side chain of the 64 th residue of the NSE/DTE motif changing into smaller(I440+A516),the catalytic activity of the enzyme can be restored to a certain extent,but the specificity of the catalytic product of the enzyme cannot be restored.However,when(Z,E)-FPP is used as a substrate,the above mutations have similar effects on the enzyme as AaBOS.3 In summary,when(E,E)-FPP is used as substrate,the synergistic change of the upstream fourth amino acid residue and the downstream 64 amino acid residue of the NSE/DTE motif in AaBOS and NtEAS has a significant different catalytic specific effect on the enzyme.However,when(Z,E)-FPP is used as a substrate,this synergistic effect has the same effect on the catalytic specificity of the enzyme.