Planta

, Volume 222, Issue 5, pp 888–898

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system

Authors

  • Martin Ruppert
    • Department of Pharmaceutical Biology, Institute of PharmacyJohannes Gutenberg-University
  • Jörn Woll
    • Department of Pharmaceutical Biology, Institute of PharmacyJohannes Gutenberg-University
  • Anatoli Giritch
    • Icon Genetics GmbHBiozentrum Halle
  • Ezzat Genady
    • Department of Pharmaceutical Biology, Institute of PharmacyJohannes Gutenberg-University
  • Xueyan Ma
    • Department of Pharmaceutical Biology, Institute of PharmacyJohannes Gutenberg-University
    • Department of Pharmaceutical Biology, Institute of PharmacyJohannes Gutenberg-University
Original Article

DOI: 10.1007/s00425-005-0031-0

Cite this article as:
Ruppert, M., Woll, J., Giritch, A. et al. Planta (2005) 222: 888. doi:10.1007/s00425-005-0031-0

Abstract

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.

Keywords

Acetylajmalan esteraseAgrobacterium-mediated viral expressionIndole alkaloid biosynthesisPurification and functional expressionRauvolfia cell suspensions

Abbreviations

GFP

Green fluorescent protein

MSH

2-Mercaptoethanol

PMSF

Phenylmethylsulfonyl fluoride

Copyright information

© Springer-Verlag 2005