Studies on diterpene cydases found in actinomycetes.

细菌二萜环化酶基因已从二萜类抗生素生产商terpentecin获得。它们的产物ORF 11和ORF 12被证实是香叶基香叶基二磷酸（GGDP）转化为与萜三烯（TTE）具有相同基本骨架的必需产物。这两种酶的功能分析也通过使用纯化的重组酶进行。ORF11产物将GGDP转化为环化中间体（二磷酸萜二烯醇，TDP），然后由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，ORF 12产物甚至与法呢基二磷酸（FDP）反应，得到三种烯属化合物，其除了链长之外具有与由GGDP形成的那些相同的结构。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，并且具有DDXXD基序的ORF12产物通过离子化引发的底物与烯丙基碳正离子的反应随后去质子化为烯烃来完成反应。ORF12产物的动力学研究表明，ORF12产物对TDP和GGDP的亲和力高于FDP，且TDP转化为TTE的反应相对活性高于FDP。在使用TDP、GGDP或FDP作为底物的反应中，这些结果表明，由ORF 12催化的实际反应是TDP在体内转化为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.