Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.
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 (13) C- and (2) H-labelled FPP isotopomers.
Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121, Bonn, Germany.
Immo
I
Burkhardt
Institut für Chemie und Biochemie - Organische Chemie, Freie Universität Berlin, Takustraße 3, 14195, Berlin, Germany.
Thomas
T
Siemon
Institut für Chemie und Biochemie - Organische Chemie, Freie Universität Berlin, Takustraße 3, 14195, Berlin, Germany.
Matthias
M
Henrot
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143, Münster, Germany.|Institut für Angewandte Genetik und Zellbiologie, Universität für Bodenkultur Wien, Konrad-Lorenz-Straße 24/I, 3430, Tulln an der Donau, Austria.
Lena
L
Studt
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143, Münster, Germany.
Sarah
S
Rösler
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, 48143, Münster, Germany.
Bettina
B
Tudzynski
Institut für Chemie und Biochemie - Organische Chemie, Freie Universität Berlin, Takustraße 3, 14195, Berlin, Germany.
Mathias
M
Christmann
Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Gerhard-Domagk-Straße 1, 53121, Bonn, Germany. dickschat@uni-bonn.de.
Jeroen S
0000-0002-0102-0631
JS
Dickschat
D019755:Carbon-Carbon Lyases / Q000235:genetics / Q000378:metabolism*; D003500:Cyclization; D005670:Fusarium / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D045789:Sesquiterpenes, Guaiane / Q000096:biosynthesis / Q000737:chemistry
D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; C004808:farnesyl pyrophosphate; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase
Fusarium fujikuroi; NMR spectroscopy; enzyme mechanisms; isotopic labelling; terpenes