Isolation and Characterization of Two Germacrene A Synthase cDNA Clones from Chicory

Harro J. Bouwmeester ^*, Jan Kodde , Francel W.A. Verstappen , Iris G. Altug , Jan-Willem de Kraker , and T. Eelco Wallaart

Plant Research International, Business Unit Cell Cybernetics, P.O. Box 16, 6700 AA Wageningen, The Netherlands (H.J.B., J.K., F.W.A.V.); Department of Organic Chemistry, Hamburg University, D--20146 Hamburg, Germany (I.G.A.); Department of Organic Chemistry, Wageningen Agricultural University, Dreijenplein 8, 6703 HB Wageningen, The Netherlands (J.-W.d.K.); and University Centre for Pharmacy, Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands (T.E.W.)

^* Corresponding author; email: h.j.bouwmeester{at}plant.wag-ur.nl.

Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. The K_m value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.

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