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Scientific research on elucidation of terpene-cyclic reactions in the biosynthesis of bioactive microbial terpenoids.

生物活性微生物萜类化合物生物合成中萜烯循环反应阐明的科学研究。

基本信息

批准号：
13306009
负责人：
SASSA Takeshi
金额：
$ 36.03万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

(1)We isolated (+)-cyatha-3,12-diene from an erinacine-producing fungus, and confirmed its stereostructure by semi-synthesis from erinacine P. The cyathane hydrocarbon could be obtained from GGDP by the cell-free system. From a fusicoccin-mass-producing fungus we isolated (+)-δ-araneosene as the presumed bicyclic hydrocarbon intermediate. (+)-Fusicocca-3(16),10(14)-diene was also isolated and its identification suggested the presence of a new fusicoccenyl cation in the biosynthetic pathway. (2)We identified two gene clusters responsible for diterpene biosynthesis by chromosome waling from GGDP synthase genes from Phomopsis amygdali. The gene clusters led us to successful isolation and characterization of two cDNAs encoding diterpene cyclases. (3)The gene cluster for aphidicolin biosynthesis has been identified by PCR-based genome walking using reported sequence of cDNA coding the diterpene cyclase. Based on non-enzymatic cyclization under various conditions and molecular orbital calculations, cyclization mechanism for construction of aphidicolane skeleton was proposed. (4)We have cloned two diterpene cyclase genes essential for biosynthesis of terpentecin. By using recombinant enzymes, it was revealed that one enzyme converted GGDP into a cyclized intermediate (terpentedienol diphosphate), and then it was transformed into terpentetriene by the other enzyme. (5)By using the site-specific mutants and the substrate analogs, we clearly demonstrated that the steric bulk size of active site residues has an important role in determining the folding conformation of squalene substrate during the polycyclization cascade.

(1)从一种产羊尿嘧啶的真菌中分离得到(+)-cyatha-3,12-二烯，并以羊尿嘧啶p为原料半合成证实了其立体结构。我们从一种大量生产梭菌素的真菌中分离出(+)-δ-芳烃作为假定的双环烃中间体。（+）-Fusicocca-3(16),10(14)-diene也被分离出来，其鉴定表明在生物合成途径中存在一个新的fusicoccenyl阳离子。(2)我们从杏仁体的GGDP合成酶基因中鉴定出两个与染色体移位有关的二萜生物合成基因簇。这些基因簇使我们成功地分离和鉴定了编码二萜环化酶的两个cdna。(3)利用已报道的编码二萜环化酶的cDNA序列，通过PCR-based基因组行走鉴定了阿飞霉素生物合成的基因簇。基于不同条件下的非酶环化反应和分子轨道计算，提出了环化机理。(4)克隆了两个萜类环化酶基因，这些基因是萜类素生物合成所必需的。利用重组酶，发现一种酶将GGDP转化为环化中间体（萜戊二烯二磷酸），然后由另一种酶转化为萜戊三烯。(5)通过位点特异性突变体和底物类似物，我们清楚地证明了活性位点残基的空间体积大小对决定角鲨烯多环级联过程中底物的折叠构象具有重要作用。