Biochemistry, Evolutionary Engineering, and Theoretical Chemistry of Cobalamin Enzymes and Chemistry of cobalamin Composite Catalysts

钴胺素酶的生物化学、进化工程、理论化学和钴胺素复合催化剂化学

• 批准号: 13125205
• 负责人: TETSUO Toraya
• 金额: $ 25.22万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13125205/
• 关键词: vitamin B12 coenzyme; X-ray analysis; radical enzyme; diol dehydrates; theoretical calculation; density-functional-theory; composite catalyst; apoenzyme model; ビタミンB_<12>補酵素; エタノールアミンアンモニアリアーゼ; ジオールデヒドラターゼ; 立体特異性; 再活性化因子; ラジカル触媒; 自殺不活性化; ビタ

1. Science of Cobalamin Enzymes and Their Improvement by Directed Evolution (1)Recombinant glycerol dehydratase was purified, and its enzymological properties were investigated. Its X-ray structure was solved for the first time. (2)The formation of the adenine-anchored radical was crystallographically demonstrated upon illumination of the diol dehydratase-adeninylpentylcobalamin complex with visible light. (3)The structure of substrate-free form of diol dehydratase was determined. It was strongly suggested that substrate triggers the homolysis of the coenzyme Co-C bond by inducing further steric strain to the Co-C bond that had been already distorted to some extent. (4)Coenzymic activity of cobalamin analogs in which the base moiety of the coezyme B_<12> was replaced by other bases was correlated with the bulkiness of the base. It was also suggested that the nucleotide moiety is required for stabilizing radical intermediates. (5)The X-ray structures of the complexes of diol dehydratase … More with B_<12> and R-or S-enantiomer were analyzed. The stereochemical courses of all the steps of the conversion of each enantiomeric substrate to product were completely elucidated based on the X-ray structures. (6)The results obtained by this study were summarized and published as a review in Chemical Reviews, the world-most authoritative review journal in Chemistry.2. Elucidation of Enzyme Mechanism by Theoretical Chemistry (1)Each step of diol dehydratase reaction was demonstrated to be energetically feasible by density-functional-theory computations with a simple model. (2)lmportant contributions of Glu170 and His 143 in the OH group migration in diol dehydratase reaction were demonstrated by means of a quantum mechanical/molecular mechanical method with a whole enzyme model including all the atoms except hydrogens.3. Construction of B_<12> Composite Catalysts (1)Composite catalysts constructed by combination of B_<12> and apoenzyme models, such as synthetic peptide-1ipids, lipid bilayers, and inorganic polymer matrices, were demonstrated to be highly reactive in carbon-skeleton rearrangement and dehalogenation reactions. (2)An enzyme model containing B_<12> and crown compounds was synthesized, and its capability to bind K^+ was confirmed. Less

1.钴胺酶科学及其定向进化改良(1)对重组甘油脱水酶进行纯化，并对其酶学性质进行研究。首次对其X射线结构进行了解析。(2)用可见光照射二醇脱水酶-腺嘌呤基戊基钴胺络合物，结晶学上证实了腺嘌呤锚定自由基的形成。(3)确定了无底物二醇脱水酶的结构。有人强烈地认为，底物通过诱导更多的空间应变到已经在一定程度上扭曲的Co-C键，从而触发辅酶Co-C键的均裂。(4)辅酶B_lt；12&gt；的碱基被其他碱基取代的钴胺类似物的辅酶活性与碱基的膨胀性有关。也有人认为，核苷酸部分是稳定自由基中间体所必需的。(5)二醇脱水酶…络合物的X射线结构对B_&lt；12&gt；和R-或S-对映体进行了分析。根据X-射线结构，完整地解释了每个对映体底物转化为产物的所有步骤的立体化学过程。(6)对本研究的结果进行总结，并以综述的形式发表在世界最权威的化学综述期刊《化学评论》上。用理论化学阐明酶的作用机理(1)用一个简单的模型，用密度泛函理论计算证明了二醇脱水酶反应的每一步在能量上是可行的。(2)用量子力学/分子力学方法，证明了Glu170和His 143在二醇脱水酶反应中对OH基团迁移的重要贡献。B&lt；12&gt；复合催化剂的构建(1)将B&lt；12&gt；与辅酶模型(如合成肽-1脂类、脂双层和无机聚合物基质)组合而成的复合催化剂，在碳骨架重排和脱卤化反应中具有很高的活性。(2)合成了一个含有B_&lt；12&gt；和冠醚化合物的酶模型，并证实了其结合K~+的能力。较少

项目成果

Shibata, N., Masuda, J., Morimoto, Y., Yasuoka, N., Toraya, T.: "Substrate-Induced Conformational Change of a Coenzyme B 12-Dependent Enzyme : Crystal Structure of the Substrate-Free Form of Diol Dehvdratase."Biochemistry. 41(42). 12607-12617 (2002)

Shibata, N.、Masuda, J.、Morimoto, Y.、Yasuoka, N.、Toraya, T.：“辅酶 B 12 依赖性酶的底物诱导构象变化：二醇无底物形式的晶体结构

Shibata, N., Nakanishi, Y., Yamanishi, M., Yasuoka, N.Toraya, T., et al.: "Structural Rationalization for the Lack of Stereospecificity in Coenzyme B12-dependent Dial Dehydratase"J.Biol.Chem.. 278(25). 22717-22725 (2003)

Shibata, N.、Nakanishi, Y.、Yamishi, M.、Yasuoka, N.Toraya, T.等人：“辅酶 B12 依赖性二价脱水酶中缺乏立体特异性的结构合理化”J.Biol.Chem。

Shimakoshi, H., Goto, A., Tachi, Y., Naruta.Y., Hisaeda.Y.: "Synthesis and Redox Behavior of Dialkylated Dicobalt Complexes Having Two Discrete Salen Units"Tetrahedron Letters. 42. 1949-1951 (2001)

Shimakoshi, H.、Goto, A.、Tachi, Y.、Naruta.Y.、Hisaeda.Y.：“具有两个离散 Salen 单元的二烷基化二钴配合物的合成和氧化还原行为”四面体字母。

K.Sakurai, et al.: "Polysaccharide-Polynucleotide Complexes VIII. Cation-Induced Complex Formation between Polyuridylic Acid and Schizophyllan"Biopolymers. 65. 1-9 (2002)

K.Sakurai等人：“多糖-多核苷酸复合物VIII.多尿苷酸和裂褶多糖之间的阳离子诱导复合物形成”生物聚合物。

H.Shimakoshi, et al.: "Syntheses of Large-membered Macrocycles Having multiple Hydrogen Bonding Moieties"Tetrahedron Lett. 43. 8261-8264 (2002)

H.Shimakoshi等人：“具有多个氢键部分的大元大环的合成”四面体快报。