Elucidation of Reaction Mechanisms of Chain-elongation Enzymes in Isoprenoid Biosynthesis

类异戊二烯生物合成中扩链酶反应机制的阐明

• 批准号: 06403033
• 负责人: OGURA Kyozo
• 金额: $ 19.78万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06403033/
• 关键词: Isoprenoid; Biosynthesis; Farnesyl Diphosphate Synthase; Geranylgeranyl Diphosphate Synthase; Prenyltransferase; Heptaprenyl Diphosphate Synthase; Isopentenyl Diphosphate Synthase; Terpenoid

1. Site-directed mutations were introduced into thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus, and structure-reactivity relation was investigated. As a result, it has been demonstrated that Phe-220 and Gln-221 in the conserved motif FQXXDD are both essential for catalysis and that Lys-47 and Lys-183 are involved in the binding of isopentenyl diphosphate. Coupled together, these findings have led to a better understanding of the mechanism of prenyltransferase reaction.2. The genes for the two components of B.stearothermophilus heptaprenyl diphosphate synthase were cloned, sequenced, and overexpressed. The gene encoding a methyltransferease responsible for the final step in menaquinone biosynthesis was found to be located between the two genes for heptaprenyl diphosphate synthase.3. The purification of geranylgeranyl diphosphate synthase from bovine brain was achived, and the catalytic function was clarified.4. A novel type of protein modification by isoprenoid derived materials was discovered by investigating the metabolism of mevalonate in halobacterium halobium.5. Farnesol phosphokinase was found in Botryococcus braunii and its properties was disclosed.6. A new metabolic system for the conversion of dolichols and dehydrodolichols into their aldehydes was discovered.

1.对嗜热脂肪芽孢杆菌耐热法尼基二磷酸合酶进行定点突变，并研究其结构-反应关系。结果表明，保守基序 FQXXDD 中的 Phe-220 和 Gln-221 对于催化都是必需的，并且 Lys-47 和 Lys-183 参与异戊烯基二磷酸的结合。这些发现结合在一起，使人们更好地了解异戊烯基转移酶反应的机制。2．对嗜热脂肪芽胞杆菌庚烯基二磷酸合酶的两个组分的基因进行了克隆、测序和过表达。编码负责甲基萘醌生物合成最后一步的甲基转移酶的基因被发现位于庚烯基二磷酸合酶的两个基因之间。3．实现了牛脑香叶基香叶基二磷酸合酶的纯化，并阐明了其催化功能。 4．通过研究盐杆菌中甲羟戊酸的代谢，发现了一种类异戊二烯衍生材料对蛋白质的新型修饰。5．在布氏葡萄球菌中发现了金合欢醇磷酸激酶，并揭示了其性质。 6．发现了一种将多羟基化合物和脱氢多羟基化合物转化为醛的新代谢系统。

项目成果

小倉協三: "培風館" 有機化学の最近の展開(日本化学研究会編):イソプレノイドの生合成化学(印刷中), 総

小仓恭三：《百风馆》有机化学最新进展（日本化学研究会编）：类异戊二烯的生物合成化学（出版中）、综合

古山種俊: "新タンパク質応用工学第11章第4節" フジテクノシステムズ(株)(印刷予定),

Tanetoshi Furuyama：《新蛋白质应用工程第11章第4节》Fuji Techno Systems Co., Ltd.（待印刷），

T.Koyama et al.: "Significance of Phe-220 and Gln-221 in the Catalytic Mechanism of farnesyl Diphosphate Synthase of Bacillus stearothermophilus" Biochem.Biophys.Res.Commun.212. 681-686 (1995)

T.Koyama 等人：“Phe-220 和 Gln-221 在嗜热脂肪芽孢杆菌法尼基二磷酸合酶催化机制中的意义”Biochem.Biophys.Res.Commun.212。

T.Koyama et al.: "Identification of Significant Residues in the Substrate Binding Site of Farnesyl Diphosphate Synthase" Biochemistry. 35. 9533-9538 (1996)

T.Koyama 等人：“法尼基二磷酸合酶底物结合位点中重要残基的鉴定”生物化学。

K.Ogura et al.: Polyprenyl Diphosphate Synthases "Subcellular Biochemistry, Vol.28, Cholesterol : Its Functions and Metabolism in Biology and Midicine" J.R.Harris ed.Plenum Publishing Corporation, London, (in press),

K.Ogura 等人：聚异戊二烯二磷酸合成酶“亚细胞生物化学，第 28 卷，胆固醇：其在生物学和医学中的功能和代谢”J.R.Harris ed.Plenum Publishing Corporation，伦敦，（印刷中），