Bioorganic studies on the recogniton mechanism of the methyl groups of the substrate skeleton by squalene and oxidosqualene cyclases

角鲨烯和氧化角鲨烯环化酶对底物骨架甲基识别机制的生物有机研究

• 批准号: 09660111
• 负责人: HOSHINO Tsutomu
• 金额: $ 1.86万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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-09660111/
• 关键词: SQUALENE; HOPENE; THTRAHYMANOL; SITE-DIRECTED MUTAGENESIS; MARKOVNIKOV RULE; TRITERPENE CYCLASE; QW MOTIF; Alicyclobacillus acidocaldarius; site-directed mutagenesis

This research project has gained insights into the cyclization mechanism of squalene and oxidosqualene catalyzed by hopene, tetrahymanol and lanosterol synthases.Investigations from the two approaches of the substrate analogues and the enzymic chemistry have revealed the following new results :1. Overexpression of squalene-hopene cyclase in Escherichia coli Biosci. Biotechnol. Biochem. 62, pp. 407-411(1998).2. Site-directed mutagenesis experiments, targetted for tryptophan residues, revealed that the repetitive conserved alighnments, i.e. QW motifs, which has been presumed to be active sites by some workers, were not active sites and had a specific function against thermal denaturation. Biosci. Biotechnol. Biochem. 63, (1999), in press.3. Point mutations of W489F and W l 69H has led us to propose the new cyclization mechanism, that is, a ring expansion from 5- to 6-membered D-ring occur during the polycyclization. J.Chem. Soc. Chem. Commun. 2617-2618 (1998).4. The substrate analogues showed that a ring expansion reaction ocurs also at the state of C-ring formation in the case of lanosterol synthase.J.Chem. Soc. Chem. Commun. 1591-1592 (1998).5. the terminal methyl groups are critical to the correct folding of squalene substrate both for the formation of the five-membered B-ring and for the initiation of the polycyclization reaction submitted for publication.

本研究对角鲨烯和氧化角鲨烯催化环化的反应机理进行了深入的研究，从底物类似物和酶化学两个方面对角鲨烯-环化酶在大肠杆菌中的高效表达进行了研究。生物技术。生物化学。62，第407-411页(1998)。针对色氨酸残基的定点突变实验表明，重复的保守序列，即QW基序，被一些工作者认为是活性部位，但不是活性部位，具有抵抗热变性的特异性功能。比奥西。生物技术。生物化学。63，(1999)，出版社。W489F和W L 69H的点突变使我们提出了新的环化机制，即在多环化过程中发生从5元D环到6元D环的扩环。J·化学。SoC。化学。交警。2617-2618(1998).底物类似物表明，在羊毛甾醇合成酶的情况下，C-环的形成也发生了扩环反应。SoC。化学。交警。1591-1592(1998).5.末端甲基对于角鲨烯底物的正确折叠是关键的，对于形成五元B环和启动提交发表的多环化反应都是关键的。

项目成果

Tsutomu Sato, Takamasa Abe and Tsutomu Hoshino: "On the cyclization mechanism of squalene : a ring expansion process of the five-membered D-ring intermediate" J.Chem.Soc.Chem.Commun.2617-2618 (1998)

Tsutomu Sato、Takamasa Abe 和 Tsutomu Hoshino：“关于角鲨烯的环化机制：五元 D 环中间体的扩环过程” J.Chem.Soc.Chem.Commun.2617-2618 (1998)

T.Sato et al: "3.On the cyclization mechanism of squalene:a ring expansion process of the five-membered D-ring intermediate" J.Chem.Soc.Chem.Commun.2617-2618 (1998)

T.Sato等：“3.关于角鲨烯的环化机理：五元D环中间体的扩环过程” J.Chem.Soc.Chem.Commun.2617-2618 (1998)

T.Hoshino et al: "2.Further Evidence that the polycyclization reaction by oxidosqualene-lanosterol cyclase proceeds via a ring expansion of the 5-membered" J.Chem.Soc.Chem.Commun.1591-1592 (1998)

T.Hoshino 等人：“2. 氧化角鲨烯-羊毛甾醇环化酶的多环化反应通过 5 元环扩展进行的进一步证据”J.Chem.Soc.Chem.Commun.1591-1592 (1998)

Tsutomu Sato and Tsutomu Hoshino: "Kinetic Studies on the Function of All the Conserved Tryptophans Involved Inside and Outside the QW Motifs of Squalene-hopene Cyclase : Stabilizing Effect of the Protein Structure against Thermal Denaturation." Biosci.Bi

Tsutomu Sato 和 Tsutomu Hoshino：“涉及角鲨烯-霍烯环化酶 QW 基序内部和外部的所有保守色氨酸功能的动力学研究：蛋白质结构对热变性的稳定作用。”

T.Sato et al: "1.Overexpression of Squalene-Hopene Cyclase by the pET Vector in Escherichia Coli and First Identification of Tryptophan and Aspartic Acid Residues inside the QW Motif as Active" Biosci.Biotechnol.Biochem.62巻. 407-411 (1998)

T. Sato 等人：“1. 在大肠杆菌中通过 pET 载体过度表达角鲨烯-Hopene 环化酶，并首次鉴定 QW 基序内的色氨酸和天冬氨酸残基是否具有活性” Biosci.Biotechnol.Biochem.Volume 62. 407-411 (1998)