Abstract

Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the first specific step in the biosynthesis of tropane and nicotine alkaloids. Potato (Solanum tuberosum L.) contains neither nicotine nor the medicinal tropane alkaloids hyoscyamine or scopolamine, but calystegines. They are nortropane alkaloids with glycosidase inhibitory activity. Based on the assumption of calystegine formation by the tropane alkaloid pathway, PMT genes and enzymes were investigated in potato. Sprouting tubers contained both N-methylputrescine and PMT activity. Two cDNA clones coding for PMTs were obtained together with a cDNA clone for spermidine synthase (SPDS, EC 2.5.1.16). The pmt sequences resemble those from Nicotiana tabacum (85% identity) and those from tropane alkaloid plants, Atropa belladonna (80% identity) and Hyoscyamus niger (79% identity). They are less similar to SPDS of S. tuberosum (66% identity). Expression of pmt1 and spds cDNA in Escherichia coli yielded active enzymes, while pmt2 expression resulted in insoluble protein. Chimera proteins obtained by fusion of fragments of S. tuberosum pmt2 and H. niger pmt were active as PMT, if the initial part of pmt2 was used, indicating that a mutation in the terminal part of the gene caused insolubility of the enzyme. PMT1 was purified after expression in E. coli and proved to be an active N-methyltransferase without SPDS activity. The enzyme was specific for putrescine (K (M) 250 microM) and inhibited by n-butylamine and cadaverine. While spds was transcribed in all plant organs, pmt transcripts were found in small tuber sprouts only. The results confirm that in potato genes and enzymes specific for the tropane alkaloid metabolism are expressed and active.