Functional analyses of terpene-related biosynthetic enzymes and creaion of novel cyclases

萜类生物合成酶的功能分析及新型环化酶的制备

基本信息

批准号：
15380081
负责人：
HOSHINO Tsutomu
金额：
$ 7.23万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15380081/
关键词：
squalene triterpene biosynthesis Mycobacterium tuberculosis diterpene unnatural amino acid squalene Mycobacterium tuerculosis diterpene halimane hopene cation π-interaction sesquiterpne dorimane terpenoids alteration of ter

项目摘要

This project was to elucidate the function of squalene-hopene cyclase and terpene-related biosynthetic enzymes and to create a novel terpene cyclase.1.SHC (squalene-hopene cyclase)(1)final deprotonation occurs from the terminal (Z)-Me and highly polarized water acted to abstract the proton.(2)Squalene analogs having a methyl group at different positions were synthesized in order to know how the methyl group on squalene affects the polycyclization. Introduction of Me group at 7- or 11-position resulted in no reaction.(3)SHC can accept truncated analogs having C15,C20 and C25, giving sesqui, di- and sesterterpenes.(4)We first succeeded in alteration of substrate specificity. Deletion of Gly600 alters the substrate specificity into eukaryotic type cyclase specific to (3S)-2,3-oxidosqualene.(5)Protocol of the site-specific incorporation of unnatural amino acid was developed by us. Incorporation of fluorophenylalanines at positions 605 and 365 established that cation-π interaction play a critical role for the cyclization cascade.2.Diterpene synthase from Mycobacterium tuberculosis(1)Rv3377c was cloned in E.coli. With aid of chaperon, this gene product was functionally expressed and determined to be diterpene cyclase after incubating various substrates. This product has halimane skeleton and was named tuberculosinol.(2)It turned out that Rv3378c was responsible for dephosphorylating tuberculosinol-pp.

该项目旨在阐明角鲨烯-藿烯环化酶和萜烯相关生物合成酶的功能，并创建一种新型萜烯环化酶。1.SHC（角鲨烯-藿烯环化酶）（1）最终的去质子化发生在末端（Z）-Me和高度极化的水起到夺取质子的作用。(2)合成了在不同位置具有甲基的角鲨烯类似物以了解角鲨烯上的甲基如何影响多环化。在7-或11-位引入Me基团导致不发生反应。(3)SHC可以接受具有C15、C20和C25的截短类似物，得到倍半萜、二萜和倍酯萜。 4)我们首先成功地改变了底物特异性，Gly600的缺失将底物特异性改变为特异于真核型环化酶。 (3S)-2,3-氧化角鲨烯。(5)我们开发了非天然氨基酸位点特异性掺入的方案，在605和365位掺入氟苯丙氨酸，证实阳离子-π相互作用在环化中发挥着关键作用。级联.2.结核分枝杆菌二萜合酶(1)Rv3377c被克隆于在大肠杆菌的陪伴下，该基因产物经过各种底物的孵育后得到功能表达，并确定为二萜环化酶，该产物具有卤烷骨架，被命名为tuberculosinol。(2)结果表明，Rv3378c负责tuberculosinol-的去磷酸化。页数