33 トリテルペン合成酵素の構造と機能 : β-アミリン及びルペオール合成酵素の生成物制御機構の解析(口頭発表の部)

抄録

Triterpenoids are one of the most abundant natural products commonly occuring in plants and exhibit a wide range of structural diversity. These triterpene frameworks are believed to be biosynthesized from a common precursor 2,3-oxidosqualene by distinct triterpene synthases. In order to identify the origin of the product specificities exhibited by triterpene synthases, we have chosen β-amyrin synthase (PNY) and lupeol synthases (LUP1, OEW) for mechanistic studies. The cyclization mechanisms leading to β-amyrin and lupeol are identical up to lupenyl cation stage where proton abstraction from the methyl group results in lupeol while ring expansion and hydride shift will generate β-amyrin. To determine the polypeptide region important for the product specificity, several chimeric enzymes were constructed. Chimera 1, in which N-terminal half is PNY and C-terminal half is LUP1, produced both β-amyrin and lupeol in 3:1 ratio. In addition, minor amount of butyrospermol (4) was produced. The results from other chimeric enzymes indicated that the 80 amino acid sequence located in the second quarter from N-terminus was important for β-amyrin formation. [1,2-^<13>C_2]Acetate feeding experiment was conducted to identify from which methyl group is proton abstracted during lupeol formation. The result from LUP1 showed that the proton is abstracted from both methyl groups in non-specific manner. On the other hand, OEW exhibited specific proton abstraction from (7)-methyl group of 2,3-oxidosqualene. These results suggested the occurence of two types of lupeol synthases in nature. Furthermore, site-directed mutagenesis was carried out in order to define the amino acid residue responsible for product specificity. PNY W259L mutant gave significant amount of lupeol together with β-amyrin, while OEW L258W mutant gave exclusively β-amyrin. PNY Y261H mutant gave neither β-amyrin nor lupeol and instead it produced mixture of 5 and 6. These results suggested that Trp 259 of PNY stabilizes the oleanyl cation during β-amyrin formation, while Tyr 261 is responsible for the formation of pentacyclic triterpenes.