Fuctional Expression, Characterization, Site-directed Mutagenesis, Crystallization And 3D-Structure Of Perakine Reductase From Rauvolfia Serpentina

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 Rauvofia 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.The first part of this thesis describes the expression and characterization of PR.The enzyme was functionally expressed in Escherichia coli as the N-terminal His₆-tagged protein and was purified to homogeneity.PR displays a broad substrate acceptance, converting 17 out of 30 tested compounds with reducible carbonyl function.The compounds belong to three substrate groups:benzaldehyde derivatives,cinnamic aldehyde derivatives and monoterpenoid indole alkaloids.The enzyme has an extraordinary selectivity in the group of alkaloids.Sequence alignments define PR as a new member of the aldo-keto reductase(AKR) superfamily,exhibiting the conserved catalytic tetrad Asp52,Tyr57,Lys84,His126.Site-directed mutagenesis of each of these functional residues to an alanine residue results in＞97.8%loss of enzyme activity,in compounds for each substrate group. The second part of this thesis reports on crystallization of native PR,co-crystallization 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 pH 7.0 buffer and 25%PEG 4000 as precipitant,enzyme concentration 4.5-5.0 mg/ml.Crystals belong to space group C222₁ and diffract to 2.30(?),with unit-cell parameters a = 58.796(?),b = 93.042(?),c = 142.997(?),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 2.30(?) resolution.R and R_free of PR structure model are 19.0%and 24.7%.PR folds as the(α/β)₈ barrel(TIM-barrel).It was unable to obtain the PR-cofactor/substrate complexes by soaking and co-crystallization.Based on PR 3D-structure,mutation of functional residues(Asp52,Tyr57,Lys84,Lys126) and the 3D-structure of other AKRs-cofactor/substrate complexes,we deduced the cofactor/substrate binding site of PR and proposed its catalytic mechanism.