The Role Of N-terminal And Glycine-439in The Catalysis Of Amorpha-4,11-diene Synthaes In Artemisia Annua L

With more than55,000members identified to date in all forms of life,the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity.The most interesting question in terpenoids study is how can living creature produce such huge number of terpenoids. The critical step to explain the terpenoid complexity is to uncover the catalytic specificity mechanism of terpene synthase. In this study, Amorpha-4,11-diene synthase of Artemisia annua(ADS) was selected for N-terminal deletion and Glycine-439site-directed mutagenesis combined with bioinformatics and molecular biology research methods to reveal their role in ADS’s catalysis.After preliminary deletion of20acid of ADS N-terminal and site-directed mutagenesis of two conservative acid, Arginine-10and Tryptophan-20, we discovered that N-terminal play an important role as a whole to keep ADS’s activity, but mutation of single acid can’t change its activity.Functional analysis G439through four mutations (G439A, S,C, V) discovered that small changes (G439A and G439S) of the cavity does not affect its activity and product specificity, but larger change results in the activity greatly reduced(G439C) or lost (G439V).These findings above, lay the foundation for further studying catalytic specificity of terpene systhases, and provide new experimental evidence for the rational design of terpenes synthase to generate compounds with new structure.