The Effect Of C Terminal On The Function Of Sesquiterpene Synthase

Terpenoids are numerous,diverse in structures and commonly found in secondary metabolites of plants and microbes.With the discovery of more and more terpenoids,the study of terpene synthase is more profound,which not only helps us to understand the synthesis mechanism of terpenoids in theory and the relationship between terpenoid synthase structure and function,but also helps to further study its use in drugs,flavors and other aspects.Based on the preliminary results of this study(The 6th amino acid truncated at the C-terminus of ADS decreased its activity),three kinds of sesquiterpene synthases with different catalytic mechanism were compared by software,and the corresponding positions were truncated.The end of the retention of several amino acids to retain its original activity to explore whether this effect is universal.The specific work is as follows:1.In order to investigate the effect of C-terminal on the function of sesquiterpene cyclase,we selected the point mutation of the C-terminus of amorpha-4,11-diene synthase(ADS),and purified proteins.The catalytic specificity of the enzyme was determined by GC-FID.Km and Kcat was determined by GC method.The results showed that C1-C10 truncation did not change the catalytic specificity of the enzyme.From the enzyme kinetic parameters point of view,C1-C6 truncation activity did not change obviously.C7-C10 truncation activity decreased,and their the kinetic parameters did not use this method to determine.2.To investigate the effect of C-terminal on the function of non-cyclized sesquiterpene synthase,we selected the point mutation of the C-terminus of non-cyclized enalene synthase((E)-?-farnesene,BFS),and purified proteins.The catalytic specificity of the enzyme was determined by GC-FID.Km and Kcat was determined by GC method.The results showed that C1-C10 truncation did not change the catalytic specificity of the enzyme.From the enzyme kinetic parameters point of view,C1-C6 truncation activity did not change obviously.C7-C10 truncation activity decreased,and their the kinetic parameters did not use this method to determine.3.In order to investigate the effect of C-terminal on the function of trans sesquiterpene cyclase,we selected the point mutations in the C-terminus of 5-epi-aristolochene synthase(EAS),and purified proteins.The catalytic specificity of the enzyme was determined by GC-FID.Km and Kcat was determined by GC method.The results showed that C1-C10 truncation did not change the catalytic specificity of the enzyme.From the enzyme kinetic parameters point of view,C1-C6 truncation activity did not change obviously.C7-C10 truncation activity decreased,and their the kinetic parameters did not use this method to determine.Based on the structural information of the three sesquiterpene synthases,we found that the truncation of 1-10 amino acids at the C-terminus of the three enzymes did not change the product.Compared with wild type,the amino acid on the ?-helix would decrease the enzyme activity,but the amino acid other than the alpha helix would not obviously change the activity.