INVESTIGATION ON THE SQUALENE CYCLASE ON THE BASES OF ORGANIC CHEMISTRY AND MOLECULAR BIOLOGY

基于有机化学和分子生物学的角鲨烯环化酶研究

• 批准号: 11660104
• 负责人: HOSHINO Tsutomu
• 金额: $ 2.05万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660104/
• 关键词: squalene; oxidosqualene; triterpene; hopene; lanosterol; cyclase; site-directed mutagenesis; Alicyclobacillus acidocaldarius

This investigation was carried out to gain insight into the squalene cyclization mechanism by using the substrate analogs and by site-directed mutagenesis experiments. We established the initiation site of the cyclization and the stabilization sites as follows. The amino acid alignment of DXDDTA motif, the acidity being enhanced by His 451, is responsible for the initiation and Phe365, Phe601 and Phe605 residues stabilize the cationic intermediates. Incubation experiments of 10-desmethylsqualene analog showed the methyl group at 10-position is most critical to the completion of squalene cyclization.

通过底物类似物和定点突变实验，深入了解角鲨烯环化机制。我们确定了环化的起始位点和稳定化位点如下。DXDDTA基序的氨基酸排列，酸度被His 451增强，负责起始和Phe 365，Phe 601和Phe 605残基稳定阳离子中间体。10-去甲基角鲨烯类似物的孵育实验表明，10-位上的甲基对角鲨烯环化的完成最为关键。

项目成果

T.Hoshino, T.Abe and M.Kouda.: "Unnatural natural triterpenes produced by altering into alanine at position 261 in hopene synthase and the importance of having the appropriate bulk size at this position for directing the stereochemical destiny during the

T.Hoshino、T.Abe 和 M.Kouda.：“通过在希望烯合酶的 261 位转变为丙氨酸而产生非天然的天然三萜，以及在该位置具有适当的体积大小对于指导立体化学命运的重要性

T.Sato and T.Hoshino.: "Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis experiments : initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially c

T.Sato 和 T.Hoshino.：“通过定点诱变实验对角鲨烯-霍烯环化酶中的 DXDDTA 基序进行功能分析：多环化反应的起始位点和初始 c 碳阳离子中间体的稳定位点

T.Hoshino: "Unnatural natural triterpenes produced by altering into alanine at position 261 in hopene synthase and the importance of having the appropriate bulk size at this position for directing the stereochemical destiny during the polycyclization casc

T.Hoshino：“通过在hopene合酶的261位上转变为丙氨酸而产生非天然的天然三萜，以及在该位置上具有适当的体积大小对于在多环化casc过程中指导立体化学命运的重要性

T. Sato and T. Hoshino: "Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis"Biosci. Biotechnol. Biochem.. 63. 2189-2198 (1999)

T. Sato 和 T. Hoshino：“通过定点诱变对角鲨烯-霍烯环化酶中 DXDDTA 基序进行功能分析”Biosci。

T.Sato: "Functional analysis of the DXDDTA motif in squalene-hopene cyclase by site-directed mutagenesis experiments : initiation site of the polycyclization reaction and stabilization site of the carbocation intermediate of the initially cyclized A-ring.

T.Sato：“通过定点诱变实验对角鲨烯-藿烯环化酶中的 DXDDTA 基序进行功能分析：多环化反应的起始位点和初始环化 A 环的碳阳离子中间体的稳定位点。