Hashimoto T, Yamada Y
Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan.
Plant Physiol. 1986 Jun;81(2):619-25. doi: 10.1104/pp.81.2.619.
Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6beta-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6beta-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe(2+) ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-(14)C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO(2) from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6beta-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.
多种茄科植物的根培养物在体外生长良好，并能产生大量的托烷生物碱。黑种草培养根的无细胞提取物中存在将莨菪碱转化为6β-羟基莨菪碱的酶活性。莨菪碱6β-羟化酶被纯化了3.3倍并进行了特性鉴定。羟基化反应对莨菪碱、2-氧代戊二酸、Fe(2+)离子和分子氧有绝对需求，抗坏血酸可刺激该反应。只有莨菪碱的l-异构体可作为底物；d-莨菪碱几乎无活性。对颠茄属、曼陀罗属、澳洲茄属、黑种草属和烟草属植物的一些根、茎和愈伤组织培养物进行了羟化酶活性检测。使用[1-(14)C]-2-氧代戊二酸研究了转化反应过程中2-氧代戊二酸的脱羧作用。结果表明，2-氧代戊二酸依赖莨菪碱生成CO(2)与莨菪碱羟基化之间呈1:1的化学计量关系。因此，该酶可归类为2-氧代戊二酸依赖型双加氧酶（EC 1.14.11.-）。莨菪碱的供应和羟化酶活性都决定了生物碱产生培养物中6β-羟基莨菪碱和东莨菪碱的产量。
