Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β-hydroxylase from Brugmansia sanguinea – tuning the scopolamine production†,RSC Medicinal Chemistry

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Isolation, expression and biochemical characterization of recombinant hyoscyamine-6β-hydroxylase from Brugmansia sanguinea – tuning the scopolamine production†
RSC Medicinal Chemistry ( IF 4.1 ) Pub Date : 2018-05-02 00:00:00 , DOI: 10.1039/c8md00090e
Conrad Fischer _{1,

2,

3,

4} , Moonhyuk Kwon _{4,

5,

6,

7} , Dae-Kun Ro _{4,

5,

6,

7} , Marco J. van Belkum _{1,

2,

3,

4} , John C. Vederas _{1,

2,

3,

4}

Affiliation

Hyoscyamine-6β-hydroxylase (H6H, EC 1.14.11.11) is a plant enzyme that catalyses the last two steps in the biosynthesis of the anticholinergic drug scopolamine, i.e. the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine (anisodamine) and subsequent oxidative ring-closure to the 6,7-β-epoxide. A H6H gene homologue was isolated from the plant Brugmansia sanguinea (BsH6H) and recombinantly cloned into Escherichia coli, expressed and purified using an effective SUMO-fusion procedure. Enzymatic activity is approximately 40-fold higher for the first reaction step and the substrate affinity is comparable to other characterized H6H homologues (K_m ∼ 60 μM). Truncation of an H6H enzyme flexible N-terminal region yields an active and stable yet more compact enzyme version.

中文翻译：

几内亚Brugmansia sanguinea 的重组hyscyamine-6β-羟化酶的分离，表达和生化特征–调节东pol碱的产量†

莨菪碱-6β羟化酶（H6H，EC 1.14.11.11）是一种植物酶，其催化在抗胆碱药物东莨菪碱的生物合成，最后两个步骤即莨菪碱的羟基化6β-hydroxyhyoscyamine（山莨菪碱）和随后的氧化环合生成6,7-β-环氧化物。甲H6H基因同源物是从植物中分离的木曼陀罗属荪（BsH6H）和重组克隆到大肠杆菌中，表达并使用有效SUMO-融合过程纯化。第一步反应的酶活性高约40倍，底物亲和力可与其他特征性H6H同源物（K _m约60μM）。截短H6H酶的柔性N端区域可产生活性和稳定但更紧凑的酶版本。

更新日期：2018-05-02

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