3-D X-Ray Structure And Substrate Specificity Studies Of The Enzyme Raucaffricine Glucosidase From Rauvolfia Serpentina

Rauvolfia serpentina Benth. ex Kurz (Apocynaceae) has been used as a medicinal plant in India for thousands of years, mainly for curing fever, insanity, inflammation, and snake bites, and, nowadays hypertension, and arrhythmia. Its major active alkaloid constituents are ajmaline, ajmalicine, and reserpine. The research on enzymes involved in the biosynthesis of active compounds in plants allows better understanding and utilization of Rauvolfia, steering the metabolic flux in the direction of a desired product by pathway blocking and gene manipulation strategies, and using some of the crucial enzymes in vitro for the chemoenzymatic synthesis of complex alkaloid skeletons.This thesis focuses on the detailed investigation of the Rauvolfia enzyme, raucaffricine glucosidase (RG, EC3.2.1.125), hydrolyzing the glucoalkaloid, raucafrricine, to its aglycone, vomilenine-a central intermediate of the ajmaline biosynthetic pathway. It describes the gene expression, as well as characterization, crystallization, structure elucidation, catalytic and substrate specificity mechanisms of RG. Moreover, it investigates the inhibition of RG activity by deoxypyranosylamine type inhibitor and its mechanism. X-ray diffraction data, collected at room temperature (RT,295K) and in typical cryo-conditions were compared in parallel.RG was functionally expressed in E. coli with an N-terminal (His)6tag and was purified to homogeneity via affinity chromatography. Kinetic studies of RG revealed that its Km values towards raucaffricine and strictosidine were0.78and1.7mM, respectively. In order to clarify the3-D structure, the active center, and the ligand binding mode of RG, crystallization of RG and its inactive mutant RG-E186Q was performed by hanging drop vapor diffusion method. The enzyme-ligand complexes were obtained by soaking method. In total,8datasets were collected and solved to resolutions better than2.6A:RG, RG-glycerol, RG-glucose, RG-inhibitor structures were processed with the data collected in cryo-conditions; RG-E186Q, RG-E186Q-dihydroraucaffricine, RG-E186Q-secologanin, RG-E186Q-glucose structures were processed with data collected at RT. The overall structures of RG and its inactive mutant possess the expected (β/α)8barrel fold characteristic of glycoside hydrolase family1(GH1), as seen earlier for the closely related strictosidine glucosidase (SG). Structural comparison between RG and SG revealed that it is the "wider gate" of RG that allows strictosidine to enter its catalytic site, whereas the "slot-like" entrance of SG prohibits the access of raucaffricine. The residues Trp392in RG and Trp388in SG control the gate shape and, ultimately, the acceptance of substrates. Moreover RG-Ser390proved to direct the conformation of the crucial Trp392.3-D structures, supported by the site-directed mutations and kinetic data of RG and SG, provided a structural and catalytic explanation for the substrate specificity of RG and deeper insights into the chemistry of O-glucosidase.Inhibition constants of four deoxypyranosylamine inhibitors against RG were determined:the Ki values ranged from0.06to620μM. Structural complex of RG with N-(cyclohexylmethyl)-γ-D-gluco-1,5-deoxypyranosylamine is now available. This inhibitor anchors exclusively in the active site of RG by competition with its natural substrate, providing structural evidence for a competitive inhibition mechanism. The combined kinetic and structural information revealed the importance of both, the glycone mimic and the aglycone mimic parts of the inhibitor for inhibition efficacy.The structural comparison of data collected at RT and cryo conditions suggested that the quality of obtained structure is similar, and measurements under RT conditions provide excellent measurement reproducibility. However, minor modifications were observed in side chain conformation of Glu476, probably due to changes in temperature. These results demonstrate that X-ray measurements under more "natural" conditions yield high quality datasets with single crystals, and provide unprecedented speed (2min), potentially paving the way for the renaissance of RT data collection.