Functional Expression, Characterization, Site-directed Crystal Structure Of Perakine Reductase With NADPH

The therapeutical usage of the medicinal plant Rauvolfia serpentina (Benth. ex Kurz) (Apocynaceae) includes for instance the treatment of hypertension, fevers, snake bites and insanity. R. serpentina delivers high yields of alkaloids which belong to the monoterpenoid indole alkaloids family. The major constituents of the plant root are reserpine, ajmalicine and ajmaline. Elucidation of the metabolic network allows a better understanding and utilizing this plant, 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 the R. serpentina enzyme perakine reductase (PR) which catalyzes an NADPH-dependent conversion from perakine to raucaffrinoline, a side-branch of biosynthetic pathway of the alkaloid ajmaline. The thesis includes the expression, characterization, crystallization and 3D-structure elucidation of PR and its mutant.In previous studies, sequence alignments define PR as a new member of the aldo-keto reductase (AKR) superfamily, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. We made site-directed mutants, In order to get complex structure, we made site-directed mutants and did research on it.The second part of this thesis reports on crystallization of native PR, cocrystallization of PR enzyme-cofactor/substrate complexes and elucidation of PR 3D-structure. After heterologous expression in E. coli cells, the best crystals of the methylated (His)6-PR native were obtained by the hanging-drop vapor-diffusion technique at 20℃with 0.1 M sodium citrate pH7.0 buffer and 25% PEG4000 as precipitant, enzyme concentration 4.5-5.0 mg/ml. Crystals belong to space group C2221 and diffract to 2.30 A, with unit-cell parameters a= 58.796 A, b= 93.042 A, c= 142.997 A,α=β=γ=90°, one asymmetric unit cell contains one molecular.3D-structure solution was attempted by molecular replacement (MR). PR 3D-structure has been determined to 2.30A resolution. Rwork and Rfree of PR structure model are 19.0% and 24.7%. PR folds as theα8/β6 barrel (TIM-barrel) different with other AKR. It was unable to obtain the PR-cofactor/substrate complexes by soaking and cocrystallization, so we made the mutation of PR, then we got the PR-cofactor complexes. Based on PR-cofactor 3D-structure, we know the cofactor binding site of PR and conclude its catalytic mechanism.