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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.

本课题旨在阐明角鲨烯-藿烯环化酶和萜烯相关生物合成酶的功能，并合成一种新的萜烯环化酶。角鲨烯-藿烯环化酶（SHC）(1)最终去质子化发生在末端(Z)-Me和高极化水的作用下提取质子。(2)合成了不同位置有甲基的角鲨烯类似物，以了解甲基对角鲨烯多环化的影响。在7位或11位引入Me组无反应。(3)SHC可以接受具有C15、C20和C25的截断类似物，得到倍萜类、二萜和二萜。(4)我们首先成功地改变了底物特异性。Gly600的缺失改变了底物特异性，使其成为真核型环化酶对(3S)-2,3-氧化角鲨烯的特异性。(5)我们制定了非天然氨基酸的位点特异性掺入方案。氟苯丙氨酸在605和365位的掺入证实了阳离子-π相互作用对环化级联起关键作用。在大肠杆菌中克隆了结核分枝杆菌(1)Rv3377c二萜合成酶。借助伴侣子对该基因产物进行功能表达，经不同底物孵育后确定其为二萜环化酶。该产品具有海绵状骨架，被命名为结核菌醇。(2)结果表明Rv3378c负责去磷酸化结核菌醇-pp。