Molecular evolution of the biosynthesis of necic acids from lycopsamine type pyrrolizidine alkaloids
DIETRICH OBER
Botanisches Institut
Abt. Biochemische Ökologie und Molekulare Evolution
Christian-Albrechts-Universität Kiel
Olshausenstraße 40
D-24098 Kiel
dober@bot.uni-kiel.de
http://www.uni-kiel.de/Botanik/
Pyrrolizidin Alkaloids (PAs) are esters of a necine base and a necic acid. They have shown to be an interesting system to answer questions concerning the evolutionary origin of secondary pathways. So we were able to show that homospermidine synthase, the first specific enzyme in the biosynthesis of the necine base, was invented several times independently during the angiosperm evolution. In this project we are studying an enzyme involved in the biosynthesis of a necic acid that is characteristic for pyrrolizidine alkaloids of the so-called lycopsamine type. Previous studies showed that the 13C-labelling pattern of the necic acid (2,3-dihydroxy-2-isopropylbutyrate) is identical to that of valine. Probably valine and necic acids of this type share a common biosynthesis up to the intermediate 2-oxoisovaleric acid. In valine biosynthesis this intermediate will be transaminated while the 13C-labelling patterns suggest for the necic acid a second transfer of a hydroxyethyl-TPP moiety (activated acetaldehyde), as it was catalyzed in the beginning of the valine biosynthesis by acetolactate synthase.
Based on our assumption that the specific step in necine base biosynthesis might be catalyzed by an enzyme that shares a common ancestor with acetolactate synthases involved in the biosynthesis of branched chain amino acids, we identified three classes of cDNA sequences from Eupatorium cannabinum (Asteraceae, tribe Eupatorieae) with high degree of identity to acetolactate synthases. All sequences were expressed in E. coli as insoluble inclusion bodies. Various expression strategies were tested for their ability to produce soluble and active enzyme protein to be able to characterize biochemically the newly identified proteins.
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