Molecular genetics of acetate-derived naphthylisoquinoline alkaloid biosynthesis in the liane Triphyophyllum peltatum

TONI M. KUTCHAN; SPRINGOB, KARIN
Institut für Pflanzenbiochemie
Weinberg 3
D-06120 Halle (Saale)
kutch@ipb-halle.de
kspringo@ipb-halle.de
http://www.ipb-halle.de

In members of the plant families Berberidaceae, Papaveraceae and Ranunculaceae, tetrahydrobenzylisoquinoline-derived alkaloids originate from two molecules of the primary amino acid L-tyrosine. The nitrogen atom found in the isoquinoline moiety is provided by one of the L-tyrosine molecules. However, the carbon skeleton of isoquinoline alkaloids found in species of the Ancistrocladaceae and Dioncophyllaceae originates from acetate rather than from L-tyrosine. This curious alternative evolutionary origin of isoquinoline alkaloids can best be understood by a sequence analysis and comparison of the genes encoding the biosynthetic enzymes. Naphthylisoquinoline alkaloids in the liana Triphyophyllum peltatum (Dioncophyllaceae) are derived from acetate.

Biosynthetically related compounds, acetate-derived naphthoquinones, occur not only in the Ancistrocladaceae and Dioncophyllaceae but also in several taxonomically related families, e.g. Droseraceae, Drosophyllaceae and Plumbaginaceae. It has been proposed that a polyketide synthase (PKS) provides the carbon skeleton for naphthylisoquinolines and naphthoquinones. Since both pathways appear to be closely related, the enzymology and molecular biology of the PKS was first investigated in the naphthoquinone-producing plants Drosophyllum lusitanicum and Plumbago indica. A recombinant type III PKS from Plumbago indica roots was analyzed. The enzyme was shown to catalyze the formation of a hexaketide-derived phenylpyrone, but naphthoquinones were not produced. Formation of naphthoquinones could also not be established in assays with crude protein extracts from in vitro cultures of Drosophyllum lusitanicum and Plumbago indica. These results suggest that additional enzymes or co-factors are required for the formation of the naphthalene ring. Future experiments will focus on the identification of these co-factors.

In addition, an expressed sequence tag (EST) analysis of Triphyophyllum peltatum was initiated to isolate cDNAs involved in naphthylisoquinoline biosynthesis. The main targets will be the type III PKS, the enzyme incorporating nitrogen into the isoquinoline ring and the protein that catalyzes the C-C phenol coupling reaction between the isoquinoline and the naphthalene moiety.

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