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高温法呢基二磷酸合成酶基因进行定点突变，并研究其结构与反应性的关系。结果表明，保守基序中的Phe-220和Gln-221都是催化所必需的，并且Lys-47和Lys-183参与异戊烯基二磷酸的结合。这些发现有助于我们更好地理解异戊烯基转移酶的反应机理.对嗜热脂肪B.七异戊二烯基二磷酸合酶的两个组分的基因进行克隆、测序和过表达。编码甲基转移酶的基因负责甲基萘醌生物合成的最后一步，被发现位于两个基因之间的heptaprenyl二磷酸脱氢酶。实现了牛脑中香叶基香叶基二磷酸合酶的纯化，并阐明了其催化功能.通过研究甲羟戊酸在盐生盐杆菌中的代谢，发现了一种新型的类异戊二烯衍生物对蛋白质的修饰.在布朗葡萄球菌中发现了法尼醇磷酸激酶，并对其性质进行了研究.发现了一种新的长萜醇和脱氢长萜醇转化为醛的代谢体系。

项目成果

T. Koyama et al.: "Structural and Functional Roles of the Cysteine Residues of Bacillus stearothermophilus Farnesyl Diphosphate Synthase" Biochemistry. 33. 12644-12648 (1994)

T. Koyama 等人：“嗜热脂肪芽孢杆菌法呢基二磷酸合酶的半胱氨酸残基的结构和功能作用”生物化学。

小倉協三: "酵素による炭素・炭素結合形成反応と有機合成への応用" 有機合成化学会誌, 11 (1996)

Kyozo Ogura：“酶的碳-碳键形成反应及其在有机合成中的应用”有机合成化学学会杂志，11（1996）

K.Ogura,T.Koyama: "Mechanistic Enzymology and Molecular Genetics of Chain Elongation in Isoprenoid Biosynthesis" Kodansha Scientific, 23 (1996)

K.Ogura、T.Koyama：“类异戊二烯生物合成中链延长的机械酶学和分子遗传学”讲谈社科学，23 (1996)

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），

H. Sagami et al.: "Purifation of Geranylgeranyl Diphoshate from Bovine Brain" Acta Biochim. Polonica.41. 293-302 (1994)

H. Sagami 等人：“从牛脑中纯化香叶基香叶基二磷酸酯”Acta Biochim。