Development of a New Synthetic Methods by Use of Thermostable Enzymes that catalyze C-C Bond Formation

利用催化 C-C 键形成的耐热酶开发新的合成方法

• 批准号: 05558080
• 负责人: OGURA Kyozo
• 金额: $ 5.82万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05558080/
• 关键词: Prenyltransferase; Farnesyl Diphosphate; Thermostable Enzyme; Site-directed Mutagenesis; Artificial Substrate Homologue; Synthase; Substrate Specificity; Geranyl Diphosphate; ファルネシル二リン酸; ゲラニル二リン酸; ファネシル二リン酸合成酵素; ヘプタプレニル二リン酸合成酵素; 酵素; 合成法; 不斉合

An efficient system for obtaining a large amount of a thermostable farnesyl diphosphate (FPP) synthase mutant, in which a site-directed mutation was introduced, was established. This system includes 1.Introduction of a site-directed mutagenesis, 2.Overprotection of the mutated enzyme in Escherichiacoli, 3.Purification by heat treatment followed by two kinds of chromatographies.The mutants, R295V,D296G,and H297L,which have replacements of Arg-295 with Val, Asp-296 with Gly, and His-297 with Leu, respectively, showed similar catalytic activities to that of the wild-type enzyme. However, their Km values for isopentenyl diphosphate (IPP) are approximately twice that of the wild-type. C289F,which has a replacement of Cys-289 with Phe showed a Km value for IPP 10 times larger than that of the wild-type. The mutant enzymes D224A,D224E,D225I and D228A,in which one of the Asp residues of DDXXD motif in region VI had been replaced, showed much lower activities than that of the wild-type.Detailed examination of the substrate specificities of the FPP synthase mutants described above will be carried out in order to find new enzymes that catalyze new C-C bond forming reactions that cannot be catalyzed the wild-type enzyme.

建立了一个有效的系统，用于获得大量可热稳定的Farnesyl二磷酸（FPP）合酶突变体，其中引入了位置定向的突变。该系统包括1.对位点定向的诱变的引入，2。埃斯切西科利中突变的酶的进程，3。通过热处理进行纯化，然后进行两种色谱。野生型酶的催化活性。但是，它们的二磷酸异戊烯基（IPP）的KM值约为野生型的两倍。用PHE替换Cys-289的C289F显示出IPP的KM值比野生型的值大10倍。突变酶D224A，D224E，D225I和D228A，其中已更换了VI区域的DDXXD基序中的一个ASP残基之一，显示出比野生型的活性要低得多。与野生型的尾巴相比，对FPP合成型的新car型的键盘进行了新的键合，从而发现了FPP合成型的新型均应，从而发现了新的合成型的特异性。无法催化野生型酶的反应。

项目成果

H. Sagami et al.: "Geranylgeranyl Diphosphate Synthase Catalyzing the Single Condensation between Isopentenyl Diphosphate and Farnesyl Diphosphate" J. Biochem.114. 118-121 (1993)

H. Sagami 等人：“香叶基香叶基二磷酸合成酶催化异戊烯基二磷酸和法尼基二磷酸之间的单缩合”J. Biochem.114。

K.Saiki,et al: "In vitro Heme O Synthesis by the cyoE Gene Product from Escherchia coli" J.Biol.Chem.26. 26041-26043 (1993)

K.Saiki 等人：“大肠杆菌 cyoE 基因产物的体外血红素 O 合成”J.Biol.Chem.26。

Tanetohshi Koyama: New Applied Technology on Protein Chemistry. Fuji Technology on Proten Chemistry (in press), (1995)

Tanetohshi Koyama：蛋白质化学应用新技术。

K. Saiki et al.: "In vitro Heme O Synthesis by the cyoE Gene Product from Escherichia coli" J. Biol. Chem.26. 26041-26043 (1993)

K. Saiki 等人：“来自大肠杆菌的 cyoE 基因产物的体外血红素 O 合成”J. Biol。

H.Inoue et al.: "Formation of Farnesyl Oleate and Three Other Farnesyl Fatty Acid Esters by Cell-free Extracts from Botryococcus Braunii B Race" Phytochemistry. 36. 1203-1207 (1994)

H.Inoue 等人：“通过布氏葡萄球菌 B 族的无细胞提取物形成法呢基油酸酯和其他三种法呢基脂肪酸酯”植物化学。