Studies on diterpene cydases found in actinomycetes.

融合子二萜环化酶基因是从一家二萜抗生素松油菌素生产商那里获得的。它们的产物ORF11和ORF12被证实是香叶基香叶基二磷酸(GGDP)转化为与特戊三烯具有相同基本骨架的叔戊三烯(TTE)所必需的。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(TDP)，再由ORF12产物转化为TTE。有趣的是，ORF12产物直接与GGDP反应，将GGDP转化为三种烯烃化合物。此外，ORF12产物还与法尼基二磷酸(FDP)反应生成三种烯烃化合物，除了链长外，它们与GGDP形成的化合物具有相同的结构。这些结果表明，带有DXDD基序的ORF11产物通过质子化引发的环化反应将GGDP转化为TDP，而带有DDXXD基序的ORF12产物通过底物与烯丙基碳正离子的电离引发反应以及烯烃的去质子化反应来完成反应。ORF12产物的动力学分析表明，TDP和GGDP的亲和力高于FDP，且TDP转化为TTE的反应相对活性较高。在以TDP、GGDP或FDP为底物的反应中最高。这些结果表明，ORF12在体内催化的实际反应是TDP转化为TTE。

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.