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Metabolism and Enzymology

Putrescine N-Methyltransferase in Cultured Roots of Hyoscyamus albus¹

n-Butylamine as a Potent Inhibitor of the Transferase both in Vitro and in Vivo

Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine, followed by its methylation by putrescine N-methyltransferase (PMT; EC 2.1.1.53). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus that contain hyoscyamine as the main alkaloid. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi-bi mechanism, in which the K_m values for putrescine and S-adenosyl-L-methionine are 277 and 203 µM, respectively, and the K_i value for S-adenosyl-L-homocysteine is 110 µM. PMT efficiently N-methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT, among which n-butylamine, cyclohexylamine, and exo-2-aminonorbornane were most inhibitory, with respective K_i values of 11.0, 9.1, and 10.0 µM. When n-butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine, and pseudotropine) drastically decreased at 1 mM of the exogenous monoamine, and the hyoscyamine content decreased by 52% at 6 mM, whereas the contents of 6-hydroxyhyoscyamine and scopolamine did not change. Free and conjugated forms of polyamines were also measured. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool), and the spermine content also increased slightly, whereas the spermidine content decreased slightly. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. Similar results were also obtained in root cultures of Datura stramonium. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.

³ Present address: Nihon CIBA GEIGY Co., Ltd., 10-66, Miyukicyo, Takarazuka 665, Japan.

¹ This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (No. 2660090 to T.H.).

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