Hashimoto T, Yamada Y
Eur J Biochem. 1987 Apr 15;164(2):277-85. doi: 10.1111/j.1432-1033.1987.tb11055.x.
Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.
天仙子胺6β-羟化酶是一种依赖2-氧代戊二酸的双加氧酶，在导致东莨菪碱的生物合成途径中催化l-天仙子胺羟基化为6β-羟基天仙子胺[桥本，T.和山田，Y.（1986年）《植物生理学》81卷，619 - 625页]。该酶从黑种草的根培养物中纯化得到，纯化倍数为310倍。通过Superose 12凝胶过滤测定，该酶的平均相对分子质量为41,000，在pH 7.8时表现出最大活性。酶活性需要l-天仙子胺和2-氧代戊二酸，其各自的米氏常数分别为35微摩尔和43微摩尔。Fe2 +、过氧化氢酶和抗坏血酸等还原剂能显著激活该酶。测试的十种其他氧代酸均不能替代2-氧代戊二酸，测试的九种其他二价阳离子也不能替代Fe2 +。该酶受到EDTA、钛铁试剂以及各种氧代酸和脂肪族二羧酸的中度抑制，受到硝基蓝四氮唑以及二价阳离子Mn2 +、Co2 +、Ni2 +、Cu2 +、Zn2 +、Cd2 +和Hg2 +的强烈抑制。几种吡啶二羧酸酯和邻二羟基苯基衍生物抑制羟化酶。吡啶2,4-二羧酸酯和3,4-二羟基苯甲酸是相对于2-氧代戊二酸的竞争性抑制剂，其各自的抑制常数分别为9微摩尔和90微摩尔。几种结构与l-天仙子胺相似的生物碱在托烷部分的C-6位被该酶羟基化。该酶制剂还将东莨菪碱的假定前体6,7-脱氢天仙子胺环氧化为东莨菪碱（米氏常数10微摩尔）。这种环氧化反应所需的辅因子与羟化反应相同，并且在纯化过程中，环氧化酶活性与羟化酶活性存在于相同的组分中。根据在天仙子胺6β-羟化酶部分纯化制剂中发现的羟化酶和环氧化酶活性，讨论了东莨菪碱生物合成的两种可能途径。
