Putrescine and putrescine N-methyltransferase in the biosynthesis of tropane alkaloids in cultured roots of Hyoscyamus albus

Abstract

[1,4-(14)C]Putrescine, administered to cultured roots of Hyoscyamus albus L., was converted to spermidine and spermine as well as to conjugated forms. A substantial fraction of its radioactivity (approx. 6%) was, however, incorporated into N-methylputrescine within 4 h, followed by an increase in radioactive tropane alkaloids (mostly hyoscyamine). Incubation of the roots with DL-[5-(14)C]ornithine and L-[2,3-(3)H]arginine separately resulted in rapid incorporation of each label into free putrescine and N-methylputrescine, followed by incorporation into hyoscyamine. During active alkaloid biosynthesis in H. albus roots, scarcely any radioactivity was recovered in δ-N-methylornithine. When the roots had been treated with 5 mM DL-α-difluoromethylornithine for 1 d prior to the administration of labeled ornithine, the total uptake of radioactivity into the root cells was not reduced, even though ornithine decarboxylase was specifically inactivated and the incorporation of the label to putrescine, N-methylputrescine, hyoscyamine and scopolamine markedly reduced. These and other results (Hashimoto et al., 1989, Planta 178, 123-130) are strong indications that tropane alkaloids in H. albus root cultures are synthesized from both ornithine and arginine by way of putrescine.

Two Tropinone Reductases with Distinct Stereospecificities from Cultured Roots of Hyoscyamus niger

“…These tissues are developmentally young root tissues without secondary growth (12). Putrescine N-methyltransferase (14) and hyoscyamine 6#-hydroxylase (11), other enzymes involved in the biosynthesis of hyoscyamine and scopolamine, are also highly active in the young root tissues. Together with the classical grafting experiments between alkaloid-producing and nonproducing plants (21), and alkaloid formation in organ cultures (13), these results suggest that hyoscyamine and scopolamine are mostly synthesized in the young root cells and translocated to the aerial parts of the plant.…”

“…It should be noted that in most root cultures the activities of the TRs are generally much higher than those of other enzymes in the pathway: the specific activities of the TRs are in the range of 1 to 1200 pKat/mg of protein, whereas those of putrescine N-methyltransferase (ref. 14 and our unpublished results) and hyoscyamine 6f-hydroxylase (11) …”

“…These roots were cultured in auxin-free B5 medium (7) supplemented with 3% (w/v) sucrose. All of the other cultured roots have been maintained in our laboratory for several years or were provided by Dr. P. Bachmann and cultured as described elsewhere (14). After the plants were harvested with a suction filter at the late growth phase, the roots were immediately frozen with liquid nitrogen and kept at -200C until use.…”

Genetic Transformation for Metabolic Engineering of Tropane Alkaloids

“…Nevertheless the results obtained are as disappointing as those obtained overexpressing H6H. The first report of PMT overexpression in hairy roots was with Hyoscyamus albus (Hashimoto et al, 1989). Tracer-feeding studies with radioactive aminoacids demonstrated that putrescine is the precursor of tropane alkaloids.…”

“…It was described by Mizusaki et al (1973) in tobacco for the first time, afterwards it has also been described in Hyoscyamus sp. (Hashimoto et al, 1989, Suzuki et al, 1999. The last part of the tropane alkaloid biosynthetic pathway is the enzymatic reaction catalyzed by hyoscyamine-6β-hidroxylase (H6H), a 2-oxoglutarate dependent dioxygenase (EC 1.14.11.11, MW: 41.000 ± 1.0, optimal pH 7.8) that catalyses the hydroxylation of hyoscyamine to scopolamine in two steps.…”