Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system
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Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.
KeywordsAcetylajmalan esterase Agrobacterium-mediated viral expression Indole alkaloid biosynthesis Purification and functional expression Rauvolfia cell suspensions
Green fluorescent protein
E. Genady and J. Woll thank the Botschaft der Arabischen Republik Ägypten and the Landesgraduiertenförderung (Rheinland-Pfalz, Germany) for a grant. Dr. G. v. Schumann is acknowledged for excellent assistance in enzyme purification. We also thank Prof. F. Lottspeich, R. Mentele (Martinsried, Germany) for performing partial protein sequencing, Prof. Y. Gleba, Dr. V. Klimyuk (Halle, Germany) and Prof. T. Kutchan (Halle, Germany) for helpful suggestions and linguistic help. This research was supported by Deutsche Forschungsgemeinschaft (Bonn, Bad-Godesberg, Germany), Fonds der Chemischen Industrie (Frankfurt/Main, Germany) and Bundesministerium für Bildung und Wissenschaft (Berlin, Germany).
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