Mechanisms of Catalytic Function of Prenyl Chain Elongating Enzymes

异戊二烯基链延长酶的催化作用机制

• 批准号: 09480138
• 负责人: KOYAMA Tanetoshi
• 金额: $ 8.06万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480138/
• 关键词: prenyltransferase; prenyl chain elongation; isoprenoid biosynthesis; farnesyl diphosphate; polyprenol; undecaprenyl diphosphate; heterosubunit; gene cloning; プレニルトランスフェラーゼ; ファルネシル二リン酸; ウンデカプレニル二リン酸; イソプレン鎖延長; プレニル二リン酸

1) Molecular Cloning of Undecaprenyl Diphosphate Synthase Gene and Identification of Conserved Sequences for Z-Prenyl Chain Elongating EnzymesCloning of the gene for undecaprenyl diphosphate synthase (UPS) was carried out by developing a novel screening method to detect colonies overexpressing extra prenyltransferases in Escherichia coli cells by autoradiography, providing the first primary structure for any prenyltransferases that catalyze Z-prenyl chain elongation. The deduced primary structure of the UPS is totally different from those of E-prenyl chain elongating enzymes which have characteristic conserved regions including aspartate-rich motifs. Construction of the gene expression system which enabled overproduction of the UPS in E.coil cells was successful. Purification and crystallization of the UPS was carried out.2) Elucidation of the Function-Expressing Mechanisms of Heptaprenyl Diphosphate SynthaseHeptaprenyl diphosphate synthase (HepPS) of Bacillus subtilis is composed of t … More wo dissociable subunits, componentsand I -II, both of which are required for the catalytic function. The dynamic interaction in forming a catalytically active complex was investigated by gel filtration and immunoblotting analysis. The mechanism of this heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic water-insoluble product from soluble substrates. Photoaffinity labelling experiments by use of a benzoylphenoxy analog of the allylic substrate, farnesyl diphosphate (FPP) indicated that component I participates in binding of FPP through the hydrophobic prenyl tail and that the two components with the allylic substrate form a stable ternary complex.3) Identification of Active Sites of Prenyltransferases by Genetic Engineering Introduction of many kinds of mutations in the structural genes of FPP synthase of Bacillus stearothermophilus, HepPP synthase of B.subtilis and UPS of M.Iuteus B-P 26 was carried out to find amino acid residues that were important for thecatalyticfunction or substrate binding. Less

1)十一戊烯基二磷酸合成酶基因的分子克隆和z -戊烯基链延长酶保守序列的鉴定通过建立一种新的筛选方法，通过放射自显像技术检测大肠杆菌细胞中过量表达额外戊烯基转移酶的菌落，克隆十一戊烯基二磷酸合成酶（UPS）基因，为催化z -戊烯基链延长的任何戊烯基转移酶提供了第一个主要结构。推导出的UPS的一级结构与含有富天冬氨酸基序的e-戊烯基链延长酶的一级结构完全不同。基因表达系统的构建成功地使UPS在e.c coil细胞中过量产生。对UPS进行了提纯和结晶。2) Heptaprenyl Diphosphate synthase （Heptaprenyl Diphosphate synthase, HepPS）的功能表达机制的阐明枯草芽孢杆菌（Bacillus subtilis）的Heptaprenyl Diphosphate synthase （HepPS）由2个以上的可解离亚基组成，即组分和I -II，这两个亚基都是催化功能所必需的。通过凝胶过滤和免疫印迹分析研究了形成催化活性配合物的动态相互作用。通过假设两个亚基的结合和解离促进了从可溶性底物合成两亲性水不溶产物的催化转换，可以理解这种异质酶的机制。利用烯丙基底物的苯甲酰苯氧基类似物法尼基二磷酸（FPP）进行的光亲和标记实验表明，组分I通过疏水的戊烯基尾部参与FPP的结合，并且这两个组分与烯丙基底物形成稳定的三元配合物。通过引入嗜脂嗜热芽孢杆菌FPP合成酶、枯草芽孢杆菌HepPP合成酶和uteus b - p26的UPS结构基因的多种突变，寻找对催化功能或底物结合起重要作用的氨基酸残基。少

项目成果

Y.-W.Zhang: "Two Cistrons of the gerC Operon of Bacillus subtilis Encode the Two Subunits of Heptaprenyl Diphoshate Synthase" J.Bacteriol.179・4. 1417-1419 (1997)

Y.-W.Zhang：“枯草芽孢杆菌 gerC 操纵子的两个柠檬编码庚烯基二磷酸合成酶的两个亚基”J.Bacteriol.179・4（1997）。

T.Koyama: "Structure and Function of Polyisoprenoid Chain Elongation Enzymes Biochemical Principles and Mechanisms of Biosynthesis and Biodegradation of Polymers" Wiley-VCH,A.Steinbuchel ed., 308-315 (1998)

T.Koyama：“聚异戊二烯链延长酶的结构和功能、聚合物生物合成和生物降解的生化原理和机制”Wiley-VCH，A.Steinbuchel 编辑，308-315 (1998)

Y.-W.Zhang, T.Koyama, and K.Ogura: "Two Cistrons of the gerC Operon of Bacillus subtilis Encode the Two Subunits of Heptaprenyl Diphosphate Synthase" J.Bacteriol.179 (4). 1417-1419 (1997)

Y.-W.Zhang、T.Koyama 和 K.Ogura：“枯草芽孢杆菌 gerC 操纵子的两个顺反子编码庚烯基二磷酸合酶的两个亚基”J.Bacteriol.179 (4)。

J.Tangpakdee, Y.Tanaka, K,Ogura, T.Koyama, R.Wititsuwannakul and N.Chareonthiphakorn: "Structure of in vitro Synthesized Rubber from Fresh Bottom Fraction of Hevea Latex" Phytochemistry. 45 (2). 275-281 (1997)

J.Tangpakdee、Y.Tanaka、K、Ogura、T.Koyama、R.Wititsuwannakul 和 N.Chareonthiphakorn：“来自橡胶树乳胶新鲜底部部分的体外合成橡胶的结构”植物化学。

M.Nagaki, H.Kannari, J.Ishibashi, Y.Maki, T.Nishino, K.Ogura, and T.Koyama: "Substrate Specificity of Thermostable Farnesyl Diphosphate Synthase with Alkyl Group Homologs of Isopentenyl Diphosphate" Bioorg.Med.Chem.Lett.8 (18). 2549-2554 (1998)

M.Nagaki、H.Kannari、J.Ishibashi、Y.Maki、T.Nishino、K.Ogura 和 T.Koyama：“热稳定法尼基二磷酸合酶与异戊烯基二磷酸烷基同系物的底物特异性”Bioorg.Med.Chem