Expression,Purification,Complex3-D Structure And Meehanism Studies Of Strictosidine Glucosidase And Vinorine Svnthasein Rauvolfia Serpentina

Rauvolfia serpentine Benth. ex Kurz (Apocynaceae) is used as medicinal plant. The medicinal root constitutes ninety kinds of alkaloids and the major active constituents are Ajmaline, Ajmalicine, Reserpine and Rescinnamine. They are used for the treatment of antiarrhythmia, hypertension, sedation and analgesia. Studies of ajmaline biosynthetic pathway allows a better understanding and streering the pathway into the direction of a desired product by blocking specific enzymes with inhibitors or modifying the enzymes and reconstructing the artificial biosynthetic pathway in efficient prokaryotic systems. This thesis is carried out for the detailed investigation of two key Rauvolfia enzymes Strictosidine-O-beta-D-glucosidase (SG) and Vinorine Synthase (VS), including the expression, purification, crystallization,3D-structure elucidation and inhibitory mechanism studies of these enzymes.Relevant research on crystal structure of Strictosidine Glucosidase has been carried out in previous time. The sequence alignment and some other results show that SG belongs to Glycosyl hydrolases. Synthesis and development of potent glycosidase inhibitors are of fundamental significance e.g. for human therapy of diabetes, lysosomal storage disorders or viral infection and inhibition of those enzymes may lead to important metabolic changes.The first part of this thesis reports on the3D-structure and inhibitory mechanism of glycosidase inhibitors in complex with SG at molecular level. In order to clarify the3D-structure, binding sites and inhibitory mechanism, we try to make crystallization of complexes of SG with different deoxypyranosylamine inhibitors. Using the hanging-drop vapor-diffusion technique to produce crystals, and with soaking method, we obtained four datasets SG-inhibitor complexes, and two complexes have been resolved and analyzed. The results show a unique visual and structural recognition on binding and inhibitory action of this kind of inhibitors. Meanwhile, it provides the structural information for the design and organic synthesis of new inhibitors.VS is the first example of crystallization and preliminary X-ray diffraction analysis of an enzyme of the BAHD superfamily, but the active center and mechanism of VS still needs to be clarified in more detail. It is important to get the complex of VS with the ligands. Complex crystals of VS were prepared by enzyme modification and high-throughput screening, which need further optimization.