(+)-Germacrene A biosynthesis -: The committed step in the biosynthesis of bitter sesquiterpene lactones in chicory
(+)-Germacrene A biosynthesis -: The committed step in the biosynthesis of bitter sesquiterpene lactones in chicory
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DOI：
10.1104/pp.117.4.1381
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发表时间：
1998-08-01
期刊：
影响因子：
7.4
通讯作者：
Bouwmeester, HJ
中科院分区：
文献类型：
--
作者：
de Kraker, JW;Franssen, MCR;Bouwmeester, HJ
菊苣（Cichorium intybos L.）含有高浓度的苦味倍半萜内酯，例如愈创木内酯、乳苦素、莴苣素和8-去乙酰莴苣素。桉烷内酯和大根香叶内酯以较少量存在。它们通过甲羟戊酸-法呢基二磷酸-大根香叶烯途径的假定生物合成现已通过从菊苣根中分离（+）-大根香叶烯A合酶得到证实。这种倍半萜环化酶纯化200倍，使用阴离子交换和染料配体色谱法的组合。它具有6.6 μ M的Km值，估计分子量为54 kD，和（宽）pH最佳值约为6.7。Germacrene A，酶促产物，被证明比文献报道的稳定得多。利用热诱导科普重排生成（-）-β-榄香烯的反应，在对映选择性气相色谱柱上测定了其绝对构型。据我们所知，到目前为止，在倍半萜生物合成中，大根香叶烯A仅被报道为（假定的）酶结合的中间体，其不是被释放，而是通过由法呢基二磷酸产生它的相同酶进行另外的环化。然而，在菊苣中，吉马烯A是从萜烯环化酶中释放出来的。显然，随后的大根香叶烷骨架的氧化和/或葡糖基化以及大根香叶烯环化酶决定了是否产生愈创木烷型或桉烷型倍半萜内酯。
The leaves and especially the roots of chicory (Cichorium intybos L..) contain high concentrations of bitter sesquiterpene lactones such as the guianolides lactupicrin, lactucin, and 8-deoxylactucin. Eudesmanolides and germacranolides are present in smaller amounts. Their postulated biosynthesis through the mevalonate-farnesyl diphosphate-germacradiene pathway has now been confirmed by the isolation of a (+)-germacrene A synthase from chicory roots. This sesquiterpene cyclase was purified 200-fold using a combination of anion-exchange and dye-ligand chromatography. It has a K-m value of 6.6 mu M, an estimated molecular mass of 54 kD, and a (broad) pH optimum around 6.7. Germacrene A, the enzymatic product, proved to be much more stable than reported in literature. Its heat-induced Cope rearrangement into (-)-beta-elemene was utilized to determine its absolute configuration on an enantioselective gas chromatography column. To our knowledge, until now in sesquiterpene biosynthesis, germacrene A has only been reported as an (postulated) enzyme-bound intermediate, which, instead of being released, is subjected to additional cyclization(s) by the same enzyme that generated it from farnesyl diphosphate. However, in chicory germacrene A is released from the sesquiterpene cyclase. Apparently, subsequent oxidations and/or glucosylation of the germacrane skeleton, together with a germacrene cyclase, determine whether guaiane- or eudesmane-type sesquiterpene lactones are produced.