Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi |
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| Authors: | Immo Burkhardt Thomas Siemon Dr Matthias Henrot Dr Lena Studt Sarah Rösler Prof?Dr Bettina Tudzynski Prof?Dr Mathias Christmann Prof?Dr Jeroen S Dickschat |
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| Affiliation: | 1. Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universit?t Bonn, Bonn, Germany;2. Institut für Chemie und Biochemie – Organische Chemie, Freie Universit?t Berlin, Berlin, Germany;3. Institut für Biologie und Biotechnologie der Pflanzen, Westf?lische Wilhelms-Universit?t Münster, Münster, Germany;4. Institut für Angewandte Genetik und Zellbiologie, Universit?t für Bodenkultur Wien, Tulln an der Donau, Austria |
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| Abstract: | Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site‐directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia‐6,10(14)‐diene. The second enzyme produced eremophilene. The absolute configuration of guaia‐6,10(14)‐diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various 13C‐ and 2H‐labelled FPP isotopomers. |
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| Keywords: | enzyme mechanisms Fusarium fujikuroi isotopic labelling NMR spectroscopy terpenes |
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