Relationship between wild type and point-mutated bovine liver dihydrodiol dehydrogenase 3, DD3 in their 3D-structures and activities

野生型和点突变牛肝二氢二醇脱氢酶 3、DD3 3D 结构和活性之间的关系

• 批准号: 08672509
• 负责人: TERADA Tomoyuki
• 金额: $ 1.47万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672509/
• 关键词: dihydrodiol dehydrogenase; point mutation; 3D-structure; computer simulation; active site; activity regulation; proton relay; CD-spectrometry; 触媒機構; 補酵素結合; cDNA; ウシ肝臓; アミノ酸置換; 基質特異性; 活性発現; 点変異

I investigated the role of aldo-keto reductases which may involve in the metabolism of polycyclic aromatic hydrocarbons in animal tissues. In the course of this project, three dihydrodiol dehydrogenase [EC.1.1.2.? ] were found and purified in bovine liver cytosol. Among these enzymes, the results of substrate specificities, inhibitor sensitivities and immunological crossreactions with corresponding antibody suggested that two enzymes (DD1 and DD2) were identified with 3alpha-hydroxsteroid dehydrogenase and aldehyde reductase, respectively. However, these enzymological and immunological properties of the third enzyme (DD3) were unique among the dihydrodiol dehydrogenses which have been reported. The partial primary structure of DD3 has been reported and its important cysteine residue which may involves in the enzymatic regulation by redox balance. Based on this amino acid sequence, DNA probe was synthesized with PCR method. As a results of the plaque hybridization study, the DD3 cDNA wa … More s cloned from male adult bovine liver cDNA library. The DD3 cDNA carrying a size of 1339 bp (323 amino acids) inserted into the expression vector pET28a (pET28a-dd3). pET28a-dd3 was transformed into E.coli bacterial cells and the recombinant protein (rDD3) was expressed with IPTG.Overexpressed rDD3 was purified with one step method of nickel affinity column with high yield and quarity. According to the same method, the mutated rDD3s which were introduced their point mutations with megaprimer PCR method using mutated primers were produced in bacterial cells. The results of substrate specificities and inhibitor sensitivities of these 9 mutants (D50N,Y55F,Y55H,K84N,H117Q,H117Y,Y55F+H117Q,C145S and C193S) suggested the following roles of these amino acids in the DD3 activity. Computer simulation of 3D-structure suggests that DD3 has a typical alpha8, beta8-barrel structure and tAsp-50, Tyr-55, Lys-84, His-117 and Cys-193 may locate in the barrel and Cys-145 in the surface of structure.1. Tyrosine-55 is an essential amino acid and may play an critical role in the transfer of hydride between coenzymes and substrates (proton relay) in the DD3. In the catalytic activity of DD3, the residue which may have an ability to involve in the hydride transfer should places at position 55 in the enzyme structure.2. Both aspartate-50 and lysine-84 play an important role in the proton relay by stabilizing the intermediate (tyrosine-55, coenzyme and substrate) in the enzyme structure.3. Histidine-117 being an important residue in the active site may involve in the recognition of substrates and may not involve directly in the proton relay.4. The computer simulations and CD-spectrum studies of wild type and mutated DD3s suggested that the structural changes that affects the loss of activity.5. Cystiene-193 which places in the cleft of coenzyme binding site of DD3 plays an important role in the regulation of DD3 activity, but, cysteine-145 did not have any role in the activity because it locates in the surface of the enzyme structure.In conclusion, the study of this project disslove easily the important relationship between the enzymatic activity and the position of amino acid resdiues in the 3D-structure by the combination of site-directed mutagenesis and computer simulation of the structure of snzyme. Less

我研究了醛酮还原酶在动物组织中可能参与多环芳烃代谢的作用。在本项目过程中，三种二氢二醇脱氢酶[EC.1.1.2.?]]在牛肝细胞质中发现并纯化。其中底物特异性、抑制剂敏感性和与相应抗体的免疫交叉反应结果表明，DD1和DD2分别与3 α -羟类固醇脱氢酶和醛还原酶鉴定。然而，第三酶（DD3）的酶学和免疫学性质在已报道的二氢二醇脱氢酶中是独一无二的。据报道，DD3的部分一级结构及其重要的半胱氨酸残基可能参与氧化还原平衡的酶促调节。根据该氨基酸序列，用PCR方法合成DNA探针。通过斑块杂交研究，从雄性成年牛肝脏cDNA文库中克隆出了DD3 cDNA。全长1339bp（323个氨基酸）的DD3 cDNA插入到表达载体pET28a （pET28a- DD3）中。将pET28a-dd3转化为大肠杆菌细胞，用IPTG表达重组蛋白（rDD3）。用镍亲和柱一步法纯化过表达的rDD3，收率高，质量好。根据同样的方法，在细菌细胞中产生突变的rDD3s，并利用突变的引物用巨引物PCR方法引入其点突变。9个突变体（D50N、Y55F、Y55H、K84N、H117Q、H117Y、Y55F+H117Q、C145S和C193S）的底物特异性和抑制剂敏感性结果表明，这些氨基酸在DD3活性中的作用如下：三维结构计算机模拟表明，DD3具有典型的alpha8、beta8-筒状结构，tAsp-50、tir -55、Lys-84、His-117和Cys-193可能位于筒状结构中，Cys-145可能位于结构表面1。酪氨酸-55是一种必需氨基酸，可能在DD3中辅酶和底物之间的氢化物转移（质子接力）中起关键作用。在DD3的催化活性中，可能有能力参与氢化物转移的残基应该位于酶结构的第55位。天冬氨酸-50和赖氨酸-84都通过稳定酶结构中的中间体（酪氨酸-55、辅酶和底物）在质子接力中发挥重要作用。组氨酸-117是活性位点的重要残基，可能参与底物的识别，但可能不直接参与质子传递。对野生型和突变型DD3s的计算机模拟和cd光谱研究表明，结构变化影响了活性的丧失。半胱氨酸-193位于DD3辅酶结合位点的缝隙中，对DD3的活性起着重要的调节作用，而半胱氨酸-145由于位于酶结构的表面，对DD3的活性没有作用。综上所述，本课题的研究通过位点定向诱变与计算机模拟酶的结构相结合，很容易地消解了酶的活性与氨基酸残基在3d结构中的位置之间的重要关系。少

项目成果

寺田知行: "Cloning and expression of cDNA enccding bovine liver dihydrodiol dehyclrogenase3,DD3" Adv.Exp.Med.Biol.414. 545-553 (1997)

Tomoyuki Terada：“编码牛肝二氢二醇脱氢酶 3，DD3 的 cDNA 的克隆和表达”Adv.Exp.Med.Biol.414 (1997)。

寺田 知行: "Cloning and expression of cDNA encoding bovine liver dihydrodiol dehydrogenase 3 DD3" Advances in Experimental Medical Bidogy. 4/4. 545-553 (1997)

Tomoyuki Terada：“编码牛肝二氢二醇脱氢酶 3 DD3 的 cDNA 的克隆和表达”实验医学生物学进展 545-553 (1997)。

Terada, T., Adachi, H., Nanjo, H., Fujita, N., Takagi, T., Nishikawa, J., Imagawa, M., Nishihara, T.and Maeda, M.: "Cloning and expression of cDNA encoding bovine liver dihydrodiol dehydrogenase 3, DD3" Adv.nces in Exp.erimental Med.ical Biol.ogy. Vol.414

Terada, T.、Adachi, H.、Nanjo, H.、Fujita, N.、Takagi, T.、Nishikawa, J.、Imakawa, M.、Nishihara, T. 和 Maeda, M.：“克隆和表达

寺田知行: "Cloning expression of cDNA encoding bovine liver dihydrodiol dehydrogenase 3,DD3" Enzymology and Molecular Biology of Carbonyl Metabolism. 6. 545-553 (1997)

Tomoyuki Terada：“编码牛肝二氢二醇脱氢酶 3，DD3 的 cDNA 的克隆表达”《羰基代谢的酶学和分子生物学》6. 545-553 (1997)。