ABSTRACT
Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.
Subject(s)
Alcohol Oxidoreductases/metabolism , Plant Roots/metabolism , Plant Tubers/metabolism , Solanum tuberosum/enzymology , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/immunology , Amino Acid Sequence , Atropine/metabolism , Blotting, Western , Cloning, Molecular , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Immunohistochemistry , Microscopy, Confocal , Molecular Sequence Data , Plant Roots/genetics , Plant Tubers/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Solanum tuberosum/genetics , Tropanes/metabolismABSTRACT
Calystegines are nortropane alkaloids that are found in Solanaceae containing the classical tropane alkaloids hyoscyamine and scopolamine, and in other Solanaceae such as potato, Solanum tuberosum (L.). Calystegines are assumed to derive from the classical tropane alkaloid pathway. We isolated a cDNA from S. tuberosum with high homology to the pseudotropine-forming tropinone reductase (TRII), which presents as the first putative metabolite specific to calystegines. The equivalent amino acid sequence shows typical motifs of a short-chain dehydrogenase (SDR). The recombinant TRII protein expressed in Escherichia coli catalyzes pseudotropine formation from tropinone with a Km value, a pH optimum, substrate and co-substrate preferences similar to those reported for the TRII enzymes from other Solanaceae species. The gene is expressed in roots, tubers and aerial parts of potato. The distribution of the TRII transcript in comparison with the calystegine concentrations in the tissues suggests transport of calystegines or their precursors between potato organs.