Expression, Purification, Crystallization,Complex3-D Structure And Substrate Expanding Study Of Two Important Enzymes In Rauvolfia Serpentina

Rauvolfia serpentina Benth-. Ex Kurz (Apocynaceae) has been used as medicinal plant in India for thousands of years, mainly treating hypertension, antiarrhythmia, fevers, insanity, anti-inflammation and snake bites. The major active constituents are ajmaline, ajmalicine, reserpine and arbutin. Research on key enzymes involved in biosynthesis of activity compounds in plant allows better understanding and utilizing of Rauvolfia, steering the pathway into the direction of a desired product by pathway blocking and gene manipulation, in vitro chemoenzymatic synthesis of complex skeleton compounds. This thesis is carried out for rational substrate exploring and structure biology research of Rauvolfia enzyme Strictosidine Synthase (STRl). Besides above, The expression, purification and crystallization condition of Arbutin Synthesis (AS) in Rauvolfia is also investigated in detail.For exploring accepted novel substrates of STRl, expanding the way to various chemoenzymatic strategies, Novel substrates were rational designed basing structure information. A novel function of STRl has been detected which allows for the first time a simple enzymatic synthesis of strictosidine analogue harboring the piperazino[1,2-a]indole scaffold. Insight into the relationship between activity and conformation of substrates in STRl is provided by using state of art X-ray crystal analysis and modeling of STRl ligand complexes. Substituent effect of accepted compounds on activity loss is also discussed here. The results provide example and structural information for further STRl substrate expanding and activity mechanism research.In order to clarify the3D-structure, activity center and mechanism of reaction catalyzed by AS which perform extraordinary in substrate acceptance abitity, expression, purification and crystallization condition of AS were investigated in detail. Crystals of AS were got by regular high-throughput screening and further optimization method, which provides first example of crystal stability induced by ligand binding and opens the gate for preliminary X-ray analysis of AS.