Chimeric triterpene synthase. A possible model for multifunctional triterpene synthase
Chimeric triterpene synthase. A possible model for multifunctional triterpene synthase
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DOI：
10.1021/ja983012h
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发表时间：
1999-02-17
影响因子：
15
通讯作者：
Ebizuka, Y
中科院分区：
文献类型：
--
作者：
Kushiro, T;Shibuya, M;Ebizuka, Y
两种三萜脱氢酶，β-香树脂素合酶（EC 5.4.99.-）来自人参和羽扇豆醇合酶（EC 5.4.99.-）从拟南芥中获得的蛋白质，用于构建这两种酶之间的一系列嵌合蛋白，以研究对产物特异性重要的区域。在酵母中的功能表达和合酶产物的分析已经揭示，其中N-末端一半是β-香树脂素合酶并且C-末端一半是羽扇豆醇合酶的嵌合体1以3：1的比率产生β-香树脂素和羽扇豆醇。通过将整个序列分为四个区域，构建了两种酶的所有可能组合。其中，只有B区（从N-末端起的第二个四分之一）是β-香树脂素合酶的嵌合体7以4：1的比例产生β-香树脂素和羽扇豆醇，表明区域B在β-香树脂素形成中的重要性。通过混合PCR方法产生的另一嵌合体以1：4的比例产生β-香树脂素和羽扇豆醇，表明对产物分布重要的序列位于区域B中的80个氨基酸残基内。[1，2-C-13（2）]乙酸酯的掺入实验表明，在羽扇豆醇的形成过程中，最终的质子提取以1：1的比例从两个偕二甲基基团中的任一个发生。这是第一次证明在任何萜类化合物的生物合成过程中甲基的混乱。另一方面，在β-香树脂素形成过程中没有观察到甲基的混乱，表明羽扇烯基阳离子的异丙基必须被β-香树脂素合酶蛋白紧紧地保持。
Two triterpene synthases, beta-amyrin synthase (EC 5.4.99.-) from Panax ginseng and lupeol synthase (EC 5.4.99.-) from Arabidopsis thaliana, were used to construct a series of chimeric proteins between these two enzymes in order to investigate the region Important for product specificity. Functional expression in yeast and analysis of the synthase products have revealed that chimera 1, in which the N-terminal half is beta-amyrin synthase and the C-terminal half is lupeol synthase, produced both beta-amyrin and lupeol in a 3:1 ratio. By dividing the whole sequence into four regions, all the possible combinations of the two synthases were constructed. Among them, chimera 7, in which only region B (the second quarter from the N-terminus) is beta-amyrin synthase, produced beta-amyrin and lupeol in a 4:1 ratio, indicating the importance of region B in beta-amyrin formation. Another chimera, which was created by the mixed PCR method, produced beta-amyrin and lupeol in a 1:4 ratio, indicating that the sequence which is important for product distribution lies within 80 amino acid residues in region B. The incorporation experiment of [1,2-C-13(2)]acetate showed that, during the formation of lupeol, the final proton abstraction takes place from either of the two gem-dimethyl groups in a 1:1 ratio. This is the first demonstration of the scrambling of methyl groups during the biosynthesis of any terpenoids. On the other hand, no scrambling of methyl groups was observed during beta-amyrin formation, indicating that the isopropyl group of the lupenyl cation must be held tightly by beta-amyrin synthase protein.