Cloning of a tropane alkaloid biosynthesis gene and analysis of its expression

托烷生物碱的生物合成被认为是通过二胺腐胺进行的，然后通过腐胺N-甲基转移酶(PMT)进行甲基化。在几种茄科植物的枝根和/或培养根中发现了较高的PMT活性。从白花蛇舌兰的培养根中分离纯化了部分PMT，并对其进行了鉴定。腐胺和S-腺苷-L-蛋氨酸的Km值分别为277um和203um，对S-腺苷-L-同型半胱氨酸的KI值为110um。PMT有效地N-甲基化胺，其中至少有两个氨基被三个或四个亚甲基隔开。单胺类化合物是PMT良好的竞争性抑制剂，其中正丁胺、环己胺和外-2-氨基冰片的抑制作用最强，KI值分别为11.0、9.1和10.0微米。当加入正丁胺时，在外源单胺浓度为1 mM时，烷胺中间体(托品酮、托品和伪托品)的含量急剧下降，在6 mM时，山梨胺的含量下降了52%。正丁胺处理使腐胺含量显著增加，精胺含量略有增加，而亚精胺含量略有下降。腐胺池大小的增加(约40nmol/mg干重)足以解释总生物碱池大小的减少。这些研究进一步支持了PMT作为生物碱生物合成的第一个专一性酶的作用。

Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine,followed by its methylation by putrescine N-methyltransferase(PMT). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi bi mechanism,in which the Km values for putrescine and S-adenosyl-L-methionine are 277 muM and 203 muM,respectively,and the Ki value for S-adenosyl-L-homocysteine is 110 muM. PMT efficiently N- methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT,among which n-butylamine,cyclohexylamine and exo- 2-aminonorbornane were most inhibitory, with respective Ki values of 11.0,9.1 and 10.0 muM. When n- butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine and pseudotropine) drastically decreased at 1 mM of the exogenous monoamine, and the hyoscyamine content decreased by 52% at6 mM. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool),and the spermine content also slightly increased, whereas the spermidine content slightly decreased. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.