Research and Development of New Enzymes Catalyzing C-C Bond formation By Use of Thermostable Enzymes.

利用耐热酶催化C-C键形成的新酶的研究与开发。

基本信息

批准号：
07555252
负责人：
KOYAMA Taketoshi
金额：
$ 4.22万
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：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1. In order to construct new enzymes catalyzing novel types of C-C bond formation that is useful for application to organic synthesis of many kinds of biologically active compounds, we introduced several kinds of mutations in the structural gene of the thermostable famesyl dihosphate (FPP) synthase of Bacillus stearothermophilus and obtained the following results : (1) Identification of binding sites for an ally lic substrate such as dimethylallyl-or gernyl diphosphate and for the homoally lic substrate, isopentenyl diphosphate. (2) Identification of critical amino acid residues that reside in the catalytic site(s)of this preny ltransferase. (3) Conversion of cataly tic function synthesizing C-15 prenyl diphosphate (famesyl diphsphate) to catalyze the synthesis of longer prenyl chain length up to C-25 (farnesyl geranyl diphosphate). (4) Construction of several kinds of catalytically active FPP synthases comprising heteromeric subunits that contain amino acid replacements in the catalytically important amino acid residues in the catalytic site. (5) Construction of several kinds of chimeric enzymes consisting half of the farnesyl diphosphate synthase subunit and half of the heptaprenyl diphosphate synthase of B.Stearmophilus.2. Molecular cloning the genes encoding several kinds of bacterial preny ltransferases was carried out. The cloned preny ltransferase genes were : (1) Heptaprenyl diphosphate [(all-E)-C_<35>PP] synthase of B.stearothermophilus. (2) Hexaprenyl diphosphate [(all-E)-C_<30>PP] synthase of Micrococcus luteus B-P 26. (3) Decaprenyl diphosphate [(all-E)-C_<50>PP] synthase of paracoccus denitrificans and (4) Undecaprenyl diphosphate [(Z,E-mixed)-C_<55>PP] synthase of M.luteus B-P 26. Construction of each of these clone expression system which enables overproduction of the encoded preny ltransferase in Escherichia coli cells has also been carried out.

1. 为了构建催化新型C-C键形成的新酶，可用于多种生物活性化合物的有机合成，我们在耐热法呢基二磷酸（FPP）合酶的结构基因中引入了几种突变嗜热脂肪芽孢杆菌并获得以下结果：（1）鉴定烯丙底物（例如二甲基烯丙基-或甲酰基二磷酸）和均质底物的结合位点。 lic底物，异戊烯二磷酸。 (2)鉴定存在于该异戊二烯转移酶的催化位点的关键氨基酸残基。 (3)将合成C-15异戊二烯基二磷酸(法尼基二磷酸)的催化功能转化为催化合成更长的异戊烯基链长至C-25(法尼基香叶基二磷酸)。 (4)构建几种具有催化活性的FPP合酶，其包含在催化位点的催化重要氨基酸残基中含有氨基酸取代的异聚亚基。 (5)构建了由B.Stearmophilus.2的一半法尼基二磷酸合酶亚基和一半庚烯基二磷酸合酶组成的几种嵌合酶。对几种细菌异戊二烯转移酶的编码基因进行了分子克隆。克隆的异戊二烯转移酶基因是： (1)嗜热脂肪芽孢杆菌的二磷酸庚烯基[(全-E)-C_ 35 PP]合酶。 (2) 藤黄微球菌 B-P 26 的六戊二烯基二磷酸 [(all-E)-C_ 30>PP] 合酶。 (3) 脱氮副球菌的十异戊二烯基二磷酸 [(all-E)-C_ 50>PP] 合酶和 (4 ) 藤黄微球菌 B-P 的十一碳二烯基二磷酸 [(Z,E-混合)-C_<55>PP] 合酶26.还构建了这些克隆表达系统中的每一个，该系统能够在大肠杆菌细胞中过量产生所编码的异戊烯基转移酶。