Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties
Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties
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DOI：
10.1016/j.phytochem.2009.07.036
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发表时间：
2009-07-01
期刊：
影响因子：
3.8
通讯作者：
Meijer, Johan
中科院分区：
文献类型：
--
作者：
Andersson, Derek;Chakrabarty, Romit;Meijer, Johan
黑芥子酶（EC 3.2.1.147）是存在于十字花科植物中的β -硫代葡萄糖苷酶。这些酶通过启动次级代谢物硫代葡萄糖苷分解成有毒产物来保护植物免受病原体和害虫的侵害。植物中存在几种形式的黑芥子酶，但不同同工酶的性质和作用尚不清楚。拟南芥（Arabidopsis thaliana）模式植物似乎含有6个黑芥子酶基因（TGG1-TGG6）。为了比较不同的黑芥子酶，在毕赤酵母中克隆并过表达了叶片TGG1和根部TGG4、TGG5对应的cdna。通过亲和层析纯化his标记的重组蛋白，SDS-PAGE分析所得重组蛋白均质。确定了所有形式的芥子酶活性，并比较了对烯丙基硫代葡萄糖苷黑素的催化活性。黑芥子酶的基础活性有22倍的差异。这些酶在较宽的pH范围内具有活性，在较宽的盐浓度范围内具有较好的热稳定性和活性，但对高盐浓度敏感。黑芥子酶对抗坏血酸表现出不同的激活-抑制反应，最大活性在0.7 ~ 1 mM左右。对脱硫黑芥子酶无活性，该化合物对黑芥子酶对黑芥子素无活性。所有黑子酶也表现出o - β -葡萄糖苷酶活性，尽管效率低于黑子酶活性。讨论了植物中不同功能黑芥子酶同工酶催化性能的差异。2009爱思唯尔有限公司版权所有。
Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed. (C) 2009 Elsevier Ltd. All rights reserved.