【作者】 孙莲莉；

【导师】 赵昱； Joachim Stoeckigt；

【作者基本信息】 浙江大学 ， 药物分析学， 2008， 博士

【摘要】 夹竹桃科萝芙木属药用植物蛇根木（Rauvolfia serpentina Benth.ex Kurz）有降血压、抗心律失常、退热、抗癫、治虫蛇咬伤等功效,富含生物碱类化合物,其主要活性成分为阿吗灵、阿吗碱和利血平。对其次生代谢产物生物合成网络的研究是进一步开发利用该药用植物、调控生物代谢方向、体外异源表达活性成分的基础。本论文对催化蛇根木生物碱Perakine还原为Raucaffrinoline反应的Perakine还原酶（Perakine Reductase,PR）进行了一系列的研究,主要包括该酶的表达、生化特征、结晶和三维结构。论文的第一部分对Perakine还原酶的生物化学特征进行了研究。以pQE-2质粒为表达载体,在大肠杆菌M15中异源高效表达N端带有6个组氨酸标记的PR,并利用Ni-NTA亲和层析进行纯化。PR的底物特异性相对较低,在所测试的30个带有可还原性羰基化合物（主要为苯甲醛类衍生物、桂皮醛类衍生物及类单萜吲哚生物碱）中有17个化合物可被PR转化,但PR对类单萜吲哚类生物碱显示了很强的底物特异性。PR特异性地依赖NADPH/NADP⁺作为辅酶进行氧化还原反应的催化。氨基酸序列比对结果表明,PR为醛酮还原酶（aldo-keto reductase,AKR）超家族的新成员,具有AKR超家族成员高度保守的四个特征性氨基酸Asp52、Tyr57、Lys84、His126,分别定点突变此四个氨基酸,酶活力的丧失均在97.8%以上,证实此四个氨基酸位于活性中心,为催化残基。为进一步深入研究Perakine还原酶的三维结构、活性位点构造及催化机制,论文的第二部分对Perakine还原酶及酶与辅酶/底物复合物进行了结晶条件的摸索及X-ray三维结构的分析。经赖氨酸残基甲基化修饰后,（His）₆-PR采用气相扩散悬滴法进行结晶,优化后的结晶条件为:晶体生长温度为20℃,酶的浓度在4.5-5.0 mg/ml之间,以pH 7.0的0.1 M柠檬酸钠为结晶缓冲液,25%PEG4000为沉淀剂。所得甲基化（His）₆-PR晶体为正交晶系,属于C222₁空间群,晶胞参数为a=58.796（?）,b=93.042（?）,c=142.997（?）,α=β=γ=90°,每个不对称单元中包含一个酶分子。采用分子置换法对甲基化（His）₆-PR的三维结构进行了解析,经修正最终PR三维结构模型的R因子和R_free因子分别为19.0%和24.7%,分辨率为2.30（?）。PR折叠为（α/β）₈桶状（TIM桶状）结构。在浸泡法和共结晶都无法获得PR与辅酶/底物复合物的情况下,我们根据PR的X-ray三维结构、活性残基（Asp52、Tyr57、Lys84、His126）的定点突变结果,再结合其他AKR酶与辅酶/复合物三维结构,推测了PR辅酶与底物的结合位点及其催化机制。更多还原

【Abstract】 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.更多还原