Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.
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.
Ruppert
Martin
M
Department of Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University, Staudinger Weg 5, 55128, Mainz, Germany.
Woll
Jörn
J
Giritch
Anatoli
A
Genady
Ezzat
E
Ma
Xueyan
X
Stöckigt
Joachim
J
D000404:Ajmaline / Q000096:biosynthesis* / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004950:Esterases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017343:Genes, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011926:Rauwolfia / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000404:Ajmaline; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C053150:acetylajmaline acetylesterase