P-59 蘇苔類のジテルペン合成酵素に関する研究(ポスター発表の部)

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Diterpenes are one of biologically important pharmaceutical sources from natural origin. In higher plant (angiosperm), tricyclic and tetracyclic diterpenes are biosynthesized successively by two-step cyclization from geranylgeranyl diphosphate (GGDP) via ent-copalyl diphosphate (CDP). Each cyclization was catalyzed by two distinct diterpene cyclases (DTCs). copalyl diphosphate synthase (CPS, type-B DTC) and ent-kaurene synthase (KS. type-A DTC). Diterpene cyclases are classified into two groups, (type A and B) on the basis of cyclization mechanisms and aspartate-rich motifs. Plant CPS belongs to type B cyclases and second aspartate of DXDD motif in CPS acts as the proton donor to initiate the cyclization of GGDP. On the other hand, type A DTC has DDXXD motif and initiates the cyclyzation of CDP by an elimination of the diphosphate group and rearranges the carbon skeleton of CDP to form tricyclic and tetracyclic diterpene skeleton. Ent-kaurene is tetracyclic diterpene. and kaurane-type diterpenes exhibit various interesting biological activities. Ent-kaurene is also key intermediate in gibberellin biosynthesis, and is synthesized sequentially by type B and type A DTCs (CPS and KS) in angiosperm. Therefore. the biosynthetic genes of ent-kaurene in angiosperm have been extensively studied, and the biosynthetic pathway is thought to be conserved among plant kingdom. These suggest that the ent-kaurene synthetic genes in lower land plant would he appropriate candidates to discuss the evolution of DTCs in land plants. We have identified ent-kaurene synthetic gene, PpCPS/KS, from the moss Physcomitrella patens and have analyzed PpCPS/KS function by in vitro cell-free protein expression system. Heterologously expressed PpCPS/KS enzyme directly converted GGDP to ent-kaurene skeleton, indicating that moss PpCPS/KS is a bifunctional DTC, as well as fungus and gymnosperm DTCs. In addition, other DTCs have not found by whole genome seach in P. Patens. The liverwort Jungermannia species have been reported to produce a various bioactive kaurane diterepens. We have found that the cultured cell of J. subulata produced ent-kaurane diterepens same as wild plant, suggesting ent-kaurene synthetic gene is expressed even in the cultured cell. We cloned diterpene synthase cDNA (JsDTC1) from J. subulata cultured cells. JsDTC1 encode polypeptide of 886 amino acids and showed high similarity to PpCPS/KS. The two DXDD and DDXXD motifs were conserved.in JsDTC1, suggesting JsDTC1would be a bifunctional DTC. The functional analysis of JsDTC1 demonstrated that JsDTC1 converted GGDP to cyclic diterpenes. The identification of JsDTC1 products are now under investigation.
天然有機化合物討論会の論文
2007-08-24

P-59 蘇苔類のジテルペン合成酵素に関する研究(ポスター発表の部)

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