Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna.

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-(14)C]ornithine, L-[U-(14)C]arginine, [U-(14)C]agmaine and [1,4-(14)C]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-(14)C]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-(14)C] ornithine and L-[U-(14)C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.