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

本研究旨在阐明角鲨烯-霍烯环化酶和萜类化合物相关生物合成酶的功能，并构建一种新的萜类化合物环化酶。1. SHC（squalene-hopene cyclase）（1）末端（Z）-Me发生最终去质子化，高度极化的水起夺取质子的作用。（2）合成了在不同位置上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基团导致没有反应。（3）SHC可接受具有C15、C20和C25的截短类似物，产生倍半萜、二萜和二倍半萜。(4)We首先成功地改变了底物特异性。Gly 600的缺失将底物特异性改变为对（3S）-2，3-氧化角鲨烯特异的真核型环化酶。（5）建立了非天然氨基酸定点掺入的方法。在605和365位引入氟苯基丙氨酸，证实阳离子-π相互作用在环化级联反应中起关键作用。2.结核分枝杆菌二萜合酶（1）Rv 3377 c在大肠杆菌中克隆。在分子伴侣的辅助下，该基因产物在多种底物中进行了功能性表达，并在孵育后被确定为二萜环化酶。本品具有石碳酸骨架，命名为结核菌素。(2)It结果表明Rv 3378 c负责结核菌素-PP的去磷酸化。

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.