Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective Km values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants Vmax/Km for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.The roots of several solanaceous plants synthesize anticholinergic alkaloids such as hyoscyamine and scopolamine. The tropine moiety of these alkaloids originates from ornithine and/or arginine. In Hyoscyamus albus, the symmetric diamine putrescine derived from these amino acids is first converted to MP' by putrescine N-methyltransferase (EC 2.1.1.53) and then deaminated oxidatively to 4-methylaminobutanal which spontaneously cyclizes to give the N-methylpyrrolinium cation ( Fig. 1) (12,14,15). This oxidative deamination is catalyzed by a DAO (EC 1.4.3.6 Alkaloid-producing, cultured roots of Hyoscyamus albus contain both free putrescine and free MP in their cells (14). But to function specifically in alkaloid biosynthesis, the DAO in Hyoscyamus roots must oxidize MP more efficiently than putrescine and other diamines present. Or, these diamines 'Abbreviations: MP, N-methylputrescine; DAO, diamine oxidase. must be localized in different metabolic compartments in the cells. We here characterize the DAO from cultured Hyoscyamus roots with emphasis on its substrate specificity.
MATERIALS AND METHODS Plant MaterialsRoots of Hyoscyamus niger L. have been maintained in vitro in our laboratory since 1984 as described elsewhere (13). Prior to the experiments reported here, samples of these roots were transferred to 300-mL flasks containing 75 mL of auxinfree B5 medium (9) supplemented with 3% (w/v) sucrose, and were then cultured for 6 d. After being harvested with a suction filter, the roots were frozen immediately with liquid nitrogen and then homogenized in a Waring Blendor. The frozen homogenate was kept at -20°C until use.Pea seeds (Pisum sativum cv Alaska) were surface-sterilized and then germinated in moistened vermiculite in the dark for 8 d. The epicotyls of the etiolated pea seedlings were excised and frozen in liquid nitrogen after which they were processed in the same way as Hyoscyamus roots.All the other root, shoot and cell-suspension cultures used had been maintained in our laboratory for several years. The conditions for their culture are described elsewhere ( 14 To study substrate specificity, the ammonia produced by the DAO reaction was measured enzymatically with glutamate dehydrogenase (from beef liver; Oriental Yeast Co., Tokyo) by monitoring the decrease in NADH at 339 nm (18).When purified pea DAO preparati...