Stenzel Olaf, Teuber Michael, Dräger Birgit
Institute of Pharmaceutical Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, 06120 Halle/Saale, Germany.
Planta. 2006 Jan;223(2):200-12. doi: 10.1007/s00425-005-0077-z. Epub 2005 Aug 9.
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.
腐胺N-甲基转移酶（PMT，EC 2.1.1.53）催化托烷和烟碱生物碱生物合成中的第一步特定反应。马铃薯（Solanum tuberosum L.）既不含有烟碱，也不含有药用托烷生物碱阿托品或东莨菪碱，而是含有卡茄碱。它们是具有糖苷酶抑制活性的去甲托烷生物碱。基于通过托烷生物碱途径形成卡茄碱的假设，对马铃薯中的PMT基因和酶进行了研究。发芽的块茎中同时含有N-甲基腐胺和PMT活性。获得了两个编码PMT的cDNA克隆以及一个亚精胺合酶（SPDS，EC 2.5.1.16）的cDNA克隆。pmt序列与烟草（85%同一性）以及托烷生物碱植物颠茄（80%同一性）和黑莨菪（79%同一性）的序列相似。它们与马铃薯的SPDS的相似性较低（66%同一性）。pmt1和spds cDNA在大肠杆菌中的表达产生了活性酶，而pmt2的表达产生了不溶性蛋白质。如果使用pmt2的起始部分，通过融合马铃薯pmt2和黑莨菪pmt的片段获得的嵌合蛋白作为PMT具有活性，这表明该基因末端部分的突变导致了酶的不溶性。PMT1在大肠杆菌中表达后被纯化，证明是一种具有活性的N-甲基转移酶，没有SPDS活性。该酶对腐胺具有特异性（K（M）为250 microM），并受到正丁胺和尸胺的抑制。虽然spds在所有植物器官中都有转录，但pmt转录本仅在小块茎芽中发现。结果证实，在马铃薯中，托烷生物碱代谢特有的基因和酶是有表达且具有活性的。
