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Demonstration of Germacrene A as an Intermediate in 5-Epi-aristolochene Synthase Catalysis

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 in...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2000-03, Vol.122 (9), p.1861-1866
Main Authors: Rising, Kathleen A, Starks, Courtney M, Noel, Joseph P, Chappell, Joseph
Format: Article
Language:English
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Summary: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. Soc. 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 3H−FPP, TEAS-Y520F produces hexanes−extractable 3H with a catalytic efficiency approximately 3% that of nonmutated, recombinant TEAS. The hexanes−extractable 3H is identified as germacrene A, m/z 204, through direct GC-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.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja993584h