Abstract
The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine δ-Nmethyltransferase and δ-N-methylornithine decafboxylase were undetectable, indicating that δ-N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from dl-[5-14C]ornithine, l-[U-14C]arginine, [U-14C]agmaine and [1,4-14C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, dl-α-difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, dl-α-difluoromethylornithine. Together with the demonstration that label was incorporated from [U-14C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from dl-[ 5-14C] ornithine and l-[U-14C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.
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Abbreviations
- DFMA:
-
dl-α-difluoromethylarginine
- DFMO:
-
dl-α-difluoromethylornithine
References
Baerheim Svendsen, A., Verpoorte, R. (1983) Chromatography of alkaloids, part A: thin-layer chromatography. Elsevier, Amsterdam New York
Bey, P., Danzin, C., Jung, M. (1987) Inhibition of basic amino acid decarboxylases involved in polyamine biosynthesis. In: Inhibition of polyamine metabolism (Biological Significance and Basis for New Therapies) pp. 1–32, McCann, P.P., Pegg, A.E., Sjoerdsma, A., eds. Academic Press, New York
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Colombo, J.-P., Konarska, L. (1984) Arginase. In: Methods of enzymatic analysis, vol. 4, pp. 285–294, Bergmeyer, J., Grassl, M., eds. Verlag Chemie, Weinheim, FRG
Fuller, W.C., Gibson, M.R. (1952) The arginase-alkaloid relationship in Datura tatula L. J. Am. Pharm. Assoc. Sci. Ed. 41, 263–266
Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Nutrient requireents of suspension cultures of soybean root cells. Exp. Cell Res. 50, 151–158
Gilbertson, T.J., Leete, E. (1967) Biosynthesis of the Nicotiana alkaloids. XII. The incorporation of α- and δ-N-methylornithine into the pyrrolidine ring of nicotine. J. Am. Chem. Soc. 89, 7085–7088
Hashimoto, T., Yamada, Y., Leete, E. (1989a) Species-dependent biosynthesis of hyoscyamine. J. Am. Chem. Soc. 111, 1141–1142
Hashimoto, T., Yukimune, Y., Yamada, Y. (1989b) Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus. I. Biochemical studies. Planta 178, 123–130
Hashimoto, T., Yukimune, Y., Yamada, Y. (1989c) Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus. II. Incorporation of labelled precursors. Planta 178, 131–135
Hedges, S.H., Herbert, R.B. (1981) δ-N-Methylornithine: a natural constituent of Atropa belladonna. Phytochemistry 20, 2064–2065
Holmes, H.L. (1950) The chemistry of the tropane alkaloids. In: The alkaloids. Chemistry and physiology, vol. 1, pp. 272–375, Manske, H.F., Holmes, H.L. eds. Academic Press, New York
Kaczkowski, J., Schütte, H.R., Mothes, K. (1961) Die Rolle des Acetats in der Biosynthese der Tropanalkaloide. Biochim. Biohys. Acta 46, 588–594
Leete, E. (1980) Alkaloids derived from ornithine, lysine and nicotinic acid. In: Encylopaedia of plant physiology, N.S., vol. 8: Secondary plant products, pp. 65–91, Bell. E.A., Charlwood, B.V., eds. Springer, Berlin Heidelberg New York
Leete, E., Chedekel, M.R. (1972) The aberrant formation of (-)Nmethylanabasine from N-methyl-Δ1-piperideinium chloride in Nicotiana tabacum and N. glauca. Phytochemistry 11, 2751–2756
Leete, E., Gros, E.G., Gilbertson, T.J. (1964) Biosynthesis of the pyrrolidine ring of nicotine: feeding experiments with N15-labelled ornithine-2-C14. Tetrahedron Lett 587–592
Leistner, E., Spenser, I.D. (1973) Biosynthesis of the piperidine nucleus. Incorporation of chirally labelled [1-3H]cadaverine. J. Am. Chem. Soc. 95, 4715–4725
Mazelis, M. (1980) Amino acid catabolism. In: The biochemistry of plants. A comprehensive treatise, vol. 5, Amino acids and derivatives, pp. 542–568, Miflin, B.J., ed. Academic Press, New York
Overton, K.H. (1985) The role of leucine in terpenoid metabolism: incorporation of leucine into sesquiterpenoids and phytosterols by Andrographis tissue cultures. In: Primary and secondary metabolism of cell cultures, pp. 225–234, Neumann, K.-H., Barz, W., Reinhard, E., eds. Springer, Berlin Heidelberg New York
Payne, J., Hamill, J.D., Robins, R.J., Rhodes, M.J.C. (1987) Production of hyoscyamine by “hairy root” cultures of Datura stramonium. Planta Medica 53, 474–478
Rhodes, M.J.C., Robins, R.J., Aird, E.L.H., Payne, J., Parr, A.J., Walton, N.J. (1989) Regulation of secondary metabolism in transformed root cultures. In: Primary and secondary metabolism of plant cell cultures. II, pp. 58–72, Kurz, W.G.W., ed. Springer, Berlin Heidelberg New York
Robins, R.J., Parr, A.J., Payne, J., Walton, N.J., Rhodes, M.J.C. (1990) Factors regulating tropane-alkaloid production in a transformed root culture of a Datura Candida × D. aurea hybrid. Planta 181, 414–422
Tiburcio, A.F., Galston, A.W. (1986) Arginine decarboxylase as the source of putrescine for tobacco alkaloids. Phytochemistry 25, 107–110
Walton, N.J., Belshaw, N.J. (1988) The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures of Nicotiana hesperis. Plant Cell Rep. 7, 115–118
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We thank Miss E. Bent for valuable technical assistance and J. Eagles, K. Parsley and Dr. F. Mellon for mass-spectrometric analysis. We are grateful to Dr. A.J. Parr and Dr. M.J.C. Rhodes for helpful discussions. We are indebted to the Merrell Dow Research Institute, Cincinnati, Ohio, USA for supplying DFMA and DFMO.
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Walton, N.J., Robins, R.J. & Peerless, A.C.J. Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna . Planta 182, 136–141 (1990). https://doi.org/10.1007/BF00239995
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DOI: https://doi.org/10.1007/BF00239995