Functional Identification,Enzymatic Analysis And Preliminary Study About Mechanism Of A Eudesmane-type Sesquiterpene Synthase From Celastrus Angulatus

Celastrus angulatus Maxim is an important medicinal plant,and its secondary metabolite Celangulin V is widely used as green pesticide.In the analysis of the synthesis pathway of Celangulin V,our team speculated that Eudesmane-type sesquiterpene synthase is the key enzyme for its dihydroagarofuran skeleton.Therefore,this paper designed experiments based on a sesquiterpene synthase,namely the Ca TPS1 gene which was selected from the transcriptome data.The main content is as follows:According to bioinformatics analysis,the ORF of Ca TPS1 gene has a total length of 1662 bp,encoded 553 amino acids,and contains Rx R,DDxx D and NSE/DTE conservative motifs.It is clustered into TPS-a subfamily by phylogenetic tree analysis.Tw TPS12 gene from Tripterygium wilfordii has high homology with Ca TPS1 gene,so we constructed the two genes into the same expression system.WQ011 p RS426-Ca TPS1 and WQ011 p RS426-Tw TPS12 were constructed.The fermentation products were detected by GC-MS.It is confirmed that Ca TPS1 is an Eudesmane-type sesquiterpene synthase,and the yield of main productβ-Eudesmol is 0.697 mg/L,Tw TPS12 is also an Eudesmane-type sesquiterpene synthase,and the yield of main productα-Eudesmol is 0.602 mg/L,A prokaryotic expression vector p ET28a-Ca TPS1 was constructed and transfected into E.coli BL21（DE3）.SDS-PAGE analysis showed that the optimal induction condition was 0.2 m M IPTG 16?C culturing 24 h.The purified protein was used for vitro reaction.The results show that the optimal reaction time is 60 min,and28?C and p H 6.4 is the optimal reaction condition for Ca TPS1.K_m of Ca TPS1 is6.26±0.25μM,K_catis（1.764±0.014）×10^-3s^-1.The structure model of Ca TPS1-FPF-(Mg²⁺)₃ and Tw TPS12-FPF-(Mg²⁺)₃complex was constructed by homology modelling.These amino acids constituting the hydrophobic pockets were confirmed.Four mutant strains WQ011p RS426-Ca TPS1 W276Y,WQ011 p RS426-Ca TPS1 I300G,WQ011 p RS426-Ca TPS1Y379F and WQ011 p RS426-Ca TPS1 D448E were obtained by site-directed mutagenesis of TRP276,ILE300,TYR379 and ASP448.The types of products and yields of mutant proteins were changed.The results show that TRP276 supports the morphology of Ca TPS1 hydrophobic pocket,ILE300 is one of the reasons for the difference in the double bond position between Ca TPS1 and Tw TPS12,TYR379 is involved in the proton transfer reaction system,and ASP448 enables Ca TPS1 to bind metal ions correctly.The catalytic mechanism of Ca TPS1 from Celastrus angulatus was preliminarily explored from two aspects of function and structure,and its enzymatic properties were analyzed,which provided a theoretical basis for future research on the mechanism of Celangulin V.