Studies on diterpene cydases found in actinomycetes.

对放线菌中发现的二萜环化酶的研究。

基本信息

批准号：
14560074
负责人：
DAIRI Tohru
金额：
$ 2.18万
依托单位：
Toyama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Fubacterial diterpene cyclase genes have been doned from a diterpenoid-antibiotic, terpentecin producer. Their products, ORF11 and ORF12, were confirmed to be essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same -basic skeleton as terpentecm. Functional analyses of these two enzymes were also performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cydized intermediate (terpentedienol diphosphate, TDP), and then it was transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly reacted with GGDP and converted GGDP into three olefinic compounds. Moreover, the ORF12 product reacted even with farnesyl diphosphate (FDP) giving three olefinic compounds, which had the same structures as those formed from GGDP except for the chain-lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the reaction by an ionization-initiated reaction of substrates to an allylic carbocation followed by deprotonation to the olefin. Kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of FDP and that the relative activity of the reaction converting TDP into TTE. was highest among the reactions using TDP, GGDP, or FDP as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.

Fubacterial 二萜环化酶基因是从二萜类抗生素、萜烯生产商获得的。他们的产品 ORF11 和 ORF12 被证实对于将香叶基香叶基二磷酸 (GGDP) 转化为与萜烯具有相同基本骨架的萜三烯 (TTE) 至关重要。还使用纯化的重组酶对这两种酶进行了功能分析。 ORF11产物将GGDP转化为环化中间体（二磷酸萜二烯醇，TDP），然后通过ORF12产物转化为TTE。有趣的是，ORF12产物直接与GGDP反应，并将GGDP转化为三种烯属化合物。此外，ORF12产物甚至与法呢基二磷酸（FDP）反应，生成三种烯属化合物，其结构与GGDP形成的化合物相同，除了链长不同。这些结果表明，具有 DXDD 基序的 ORF11 产物通过质子化引发的环化将 GGDP 转化为 TDP，而具有 DDXXD 基序的 ORF12 产物通过底物电离引发的反应完成烯丙基碳正离子，然后去质子化为烯丙基碳正离子，从而完成反应。烯烃。 ORF12产物的动力学表明，对TDP和GGDP的亲和力高于FDP，并且将TDP转化为TTE的反应的相对活性。在使用 TDP、GGDP 或 FDP 作为底物的反应中最高。这些结果表明ORF12催化的实际反应是体内TDP转化为TTE。