7 高等植物におけるステロール及びトリテルペンの生合成 : オキシドスクアレン閉環酵素の精製とcDNAクローニング(口頭発表の部)

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The cyclization of 2,3-oxidosqualene to the backbones of steroid and triterpene is one of the most important and remarkable reactions found in nature. In higher plants, the cyclization of 2,3-oxidosqualene is an important branch point between sterol and triterpene biosynthesis. To understand the enzyme reaction mechanism and the regulation of the biosynthesis of steroid and triterpene, we have been studying 2,3-oxidosqualene cyclases in plants. We have tried to purify cycloartenol synthase and β-amyrin synthase from the cell suspension cultures of Rabdosia japonica and the seedlings of Pisum sativum. All of the cyclase activities were detected in membrane bound proteins and required detergents for their optimal activities. They were successfully solubilized with Triton X-100. Each solubilizedsynthase was purified with the combination of column chromatography of hydroxylapatide, DEAE-cellulose, isoelectric focusing and gel filtration. The purified cycloartenol synthases showed a single band at 54K(Rabdosia japonica) and 56K (Pisum sativum) on SDS-PAGE, respectively, while the purified β-amyrin synthases showed a single band at 28K(Rabdosia japonica) and 35K(Pisum sativum). Dammarenediol has the different skeleton from those of cycloartenol and β-amyrin and expected to be formed in a similar manner to β-amyrin until the formation of dammarenyl cation but the reaction is terminated by the addition of water before backbone rearrangement. As there has been no report on dammarenediol synthase, we have tried to detect this enzyme in the hairy root of Panax ginseng. Panax ginseng produces a number of triterpene saponins and one of the major saponins is ginsenoside R_<b-1> whose aglycone is protopanaxadiol (12β-hydroxy-dammarenediol). The microsomal preparation from the hairy root culture of this plant was able to convert [3-^3H]-2,3-oxidosqualene to (20S)-dammarenediol. The cyclization activity was highest at relatively acidic pH, and did not require any detergent. This finding was in sharp contrast to the cycloartenol and β-amyrin synthases from Rabdosia japonica and Pisum sativum all of which absolutely required detergent for their optimal activities. Several 2,3-oxidosqualene cyclases have been cloned recently from yeast, rat, human, and plant. Several oligo DNA primers based on the highly homologous regions among the known cyclases were designed for PCR amplification of cyclase cDNA from plant. PCR performed on cDNA mixture of Pisum sativum seedling using these primers gave 289 bp product which showed 79% identity to the corresponding region of Arabidopsis thaliana cycloartenol synthase cDNA. The sequence of the full length cDNA was finally obtained with 5'- and 3' RACE methods and showed 76% identity to Arabidopsis thaliana cycloartenol synthase cDNA. The identity of this cDNA as cycloartenol synthase was confirmed by functional expression in yeast.
天然有機化合物討論会の論文
1996-09-02

7 高等植物におけるステロール及びトリテルペンの生合成 : オキシドスクアレン閉環酵素の精製とcDNAクローニング(口頭発表の部)

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