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

托烷生物碱生物合成基因的克隆及表达分析

• 批准号: 02660090
• 负责人: HASHIMOTO Takashi
• 金额: $ 1.66万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660090/
• 关键词: Alkaloids; Biosynthesis; Putrescine; Cultured roots; Polyamine; 倍養根; 二次代謝産物; プトレシン; メチル基転移酵素

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.

托烷生物碱的生物合成被认为是通过二胺腐胺，然后通过腐胺N-甲基转移酶（PMT）进行甲基化。在茄科植物的分支根和/或培养根中发现了较高的PMT活性。从白天仙子的培养根中部分纯化并鉴定了PMT。PMT的初始速度研究和产物抑制模式与有序的bibi机制一致，其中腐胺和S-腺苷-L-甲硫氨酸的Km值分别为277 μ M和203 μ M，S-腺苷-L-同型半胱氨酸的Ki值为110 μ M。PMT有效地N-甲基化胺，其具有由三个或四个亚甲基分开的至少两个氨基。单胺类化合物是PMT的竞争性抑制剂，其中正丁胺、环己胺和外消旋2-氨基降冰片烷的抑制作用最强，Ki值分别为11.0、9.1和10.0 μ M。在根培养过程中，用正丁胺处理根培养物.在外源单胺浓度为1 mM时，碱胺中间产物（托品酮、托品碱和拟托品碱）的含量急剧下降，而莨菪碱的含量在6 mM时下降了52%。正丁胺处理引起腐胺含量（尤其是结合池）的大幅增加，精胺含量也略有增加，而亚精胺含量略有下降。腐胺池大小（约40 nmol/mg干重）的增加足以解释总生物碱池大小的减少。这些研究进一步支持PMT作为生物碱生物合成的第一个特异性酶的作用。

项目成果

Naruhiro Hibi: "Putrescine N-methyltransferase in cultured roots of Hyosayamus albus." Plant Physiology. 100. 826-835 (1992)

Naruhiro Hibi：“Hyosayamus albus 培养根中的腐胺 N-甲基转移酶。”

Naruhiro HIBI: "Putrescime N-methyltransferase in cultured roots of Hyoscyamus slbus." Plant Physiology. 100. 826-835 (1992)

Naruhiro HIBI：“天仙子培养根中的 Putrescime N-甲基转移酶。”

Naruhiro Hibi,et al: "Putrescine N-methyltransferase in cultured roots of Hyoscyamus albus." Plant Physiology. 100. 826-835 (1992)

Naruhiro Hibi 等人：“天仙子培养根中的腐胺 N-甲基转移酶。”