Demonstration of germacrene A as an intermediate in 5-epi-aristolochene synthase catalysis
Demonstration of germacrene A as an intermediate in 5-epi-aristolochene synthase catalysis
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DOI：
10.1021/ja993584h
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发表时间：
2000-03-08
影响因子：
15
通讯作者：
Chappell, J
中科院分区：
文献类型：
--
作者：
Rising, KA;Starks, CM;Chappell, J
倍半萜合酶是一类酶，可通过替代途径催化法尼基焦磷酸 (FPP) 环化，产生各种环状倍半萜产物。已提出由其中几种酶（包括烟草 5-表马兜铃烯合酶 (TEAS)）的催化作用，包括形成作为稳定中间体的吉甘烯 A。在正常催化过程中，倍半萜烯合酶活性位点均不会释放吉甲烯 A 或任何其他中间体。支持吉菌烯 A 中介作用的证据源自对来自土曲霉和罗氏青霉的马兜铃烯合酶的研究（Cane, D. E. Chem. Rev. 1990, 90, 1089-1103 和其中的参考文献。 Cane, D. E. ；Bryant, C. J. Am. Chem. Sec. 1994, 116, 12063-12064. Cane, D. E.; Y. S. J. 化学学会 1996, 118, 10037-10040。然而，直到目前对 TEAS 的研究，这种假定的中间体的形成从未被直接证明。 TEAS 催化 FPP 环化为 5-表马兜铃烯，这是烟草植物抗毒素卡西迪醇的前体。基于 TEAS 的三维结构，提出了 TEAS 催化的详细机制，其中包括预测 Y520 的质子捐赠负责将 Geracrene A 活化为桉烷阳离子 (Starks, C. M.; Back, K.; Chappell, J.; Noel, J.P. Science 1997, 277, 1815-1820)。在本研究中，通过定点诱变将 Y520F 点突变引入 TEAS (TEAS-Y520F)。在 H-3-FPP 存在的情况下，TEAS-Y520F 产生可己烷萃取的 H-3，其催化效率约为未突变重组 TEAS 的 3%。通过与真实样品的直接 CC-MS 比较，己烷可提取的 3H 被鉴定为germacrene A（mit 204）。这一观察结果证实了吉甘烯 A 在 TEAS 催化中的中介作用，支持了多种其他倍半萜合酶产生吉甘烯 A 的假设，并且还证实了 Y520 在 TEAS 催化中的拟议作用。
Sesquiterpene synthases are a family of enzymes that catalyze farnesyl pyrophosphate (FPP) cyclization via alternative pathways to produce a variety of cyclic sesquiterpene products. Catalysis by several of these enzymes, including tobacco 5-epi-aristolochene synthase (TEAS), has been proposed to include the formation of germacrene A as a stable intermediate. Neither germacrene A nor any other intermediate is released from sesquiterpene synthase active sites during normal catalysis. Evidence to support the intermediacy of germacrene A has been derived from investigations of aristolochene synthases from Aspergillus terreus and Penicillium roquefortii (Cane, D. E. Chem. Rev. 1990, 90, 1089-1103 and references therein. Cane, D. E.; Bryant, C. J. Am. Chem. Sec. 1994, 116, 12063-12064. Cane, D. E.; Tsantrizos, Y. S. J. Am. Chem. Soc. 1996, 118, 10037-10040). However, until the present investigations of TEAS, formation of this postulated intermediate has never been directly demonstrated. TEAS catalyzes the cyclization of FPP to 5-epi-aristolochene, a precursor of a tobacco phytoalexin, capsidiol. Based upon the three-dimensional structure of TEAS, a detailed mechanism has been proposed for TEAS catalysis that includes the prediction that proton donation by Y520 is responsible for the activation of germacrene A to a eudesmane cation (Starks, C. M.; Back, K.; Chappell, J.; Noel, J.P. Science 1997, 277, 1815-1820). In the present investigation, a Y520F point mutation is introduced into TEAS (TEAS-Y520F) by site-directed mutagenesis. In the presence of H-3-FPP, TEAS-Y520F produces hexanes-extractable H-3 With a catalytic efficiency approximately 3% that of nonmutated, recombinant TEAS. The hexanes-extractable 3H is identified as germacrene A, mit 204, through direct CC-MS comparison to an authentic sample. This observation confirms the intermediacy of germacrene A in TEAS catalysis, supports the postulated production of germacrene A by a variety of other sesquiterpene synthases, and also confirms the proposed role of Y520 in TEAS catalysis.