Expression, Purification, Crystallization, 3-D Structure And Mechanism Study Of Three Important Enzymes During Ajmaline Biosynthesis Pathway In Rauvolfia Serpentina

Rauvolfia serpentina Benth. ex Kurz (Apocynaceae) is used as medicinal plant for thousands of years, including the treatment of hypertension, antiarrhythmia, fevers, insanity and snake bites. The major constituents are ajmaline, ajmalicine and reserpine. Elucidation of ajmaline biosynthetic pathway allows a better understanding and utilizing of Rauvolfia, steering the pathway into the direction of a desired product by blocking specific enzymes with inhibitors or knocking out the corresponding cDNA, and reconstructing the artificial biosynthetic pathway in efficient prokaryotic systems. This thesis is carried out for the detailed investigation of three Rauvofia enzymes Polyneuridine Aldehyde Esterase (PNAE), Vinorine Synthase (VS) and Strictosidine Synthase (STR1), including the expression, purification, crystallization,3D-structure elucidation and mechanism study of these three enzymes.In order to clarify the 3D-structure, active center and mechanism of the reaction catalyzed by PNAE, we try to make crystallization of PNAE knock-out mutant, and PNAE knock-out mutant-substrate complexes. Using the hanging-drop vapor-diffusion technique to produce crystals, and with soaking method, we got PNAE-product complex, based on PNAE 3D-structure, product binding site of PNAE, we are able to clarify its catalytic mechanism, and structure-based design of PNAE is possible now.3D-structure of VS was already known, but the active center and mechanism of VS still needs to be clarified, it's very important to get the complex of VS. We try to make cocrystallization and soaking, process the X-ray data, unfortunately can not get complex so far.Since there was a major part of inclusion bodies formed during STR1 cDNA expression, it's a big problem for large scale using. In this thesis, we build new expression systems, compared the expression amount and activity between those new expression systems. Finally we chose one system whose expression amount is two times as the old one with the same activity. And this expression system can be used for large scale expression and for alkaloids library building.