Bouwmeester Harro J, Kodde Jan, Verstappen Francel W A, Altug Iris G, de Kraker Jan-Willem, Wallaart T Eelco
Plant Research International, Business Unit Cell Cybernetics, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Plant Physiol. 2002 May;129(1):134-44. doi: 10.1104/pp.001024.
Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K(m) value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.
最近研究表明，菊苣（Cichorium intybus）中的倍半萜内酯源自一种常见的倍半萜中间体，即（+）-吉马烯A。吉马烯A备受关注，因为它在倍半萜内酯生物合成中起关键作用，并且是多种植物抗毒素生物合成过程中的一种酶结合中间体。我们使用简并引物通过聚合酶链反应，从菊苣中分离出了两种氨基酸序列一致性达72%的倍半萜合酶。在大肠杆菌中对这些基因进行异源表达，结果表明它们都仅催化（+）-吉马烯A的形成。据我们所知，这是关于分离编码（+）-吉马烯A合酶（GAS）基因的首次报道。Northern分析表明，这两个基因在所有测试的菊苣组织中均有表达，不过表达水平有所不同。从菊苣头中分离并部分纯化蛋白质，证实存在两种GAS蛋白。在MonoQ柱上，这些蛋白质与两种异源产生的蛋白质共洗脱。大肠杆菌中产生的其中一种蛋白质的米氏常数（K(m)）值、最适pH值和MonoQ洗脱体积，与最近从菊苣根中纯化得到的GAS蛋白所报道的值相似。最后，对推导得到的两个氨基酸序列进行建模，并将所得的蛋白质模型与烟草（Nicotiana tabacum）5-表-马兜铃烯合酶的晶体结构进行比较，该合酶在生成5-表-马兜铃烯的过程中，会形成吉马烯A作为酶结合中间体。本文还讨论了一些氨基酸在倍半萜合酶产物特异性中可能发挥的作用。
