Institute of Pharmaceutical Biology, Technische Universität Braunschweig, Mendelssohnstr. 1, D-38106 Braunschweig, Germany.
Phytochemistry. 2011 Oct;72(14-15):1699-709. doi: 10.1016/j.phytochem.2011.06.013. Epub 2011 Jul 21.
Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate-myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.
硫代葡萄糖苷，一种在芸薹属植物中发现的氨基酸衍生的硫代葡萄糖苷，是研究最多的植物次生代谢物之一。与黑芥子硫苷酶和被称为特异性蛋白的补充蛋白一起，它们形成了硫代葡萄糖苷-黑芥子硫苷酶系统，当组织受到损伤时，会产生许多生物活性的硫代葡萄糖苷分解产物，如异硫氰酸酯、硫代乙腈和有机硫氰酸盐，参与植物防御。虽然异硫氰酸酯是黑芥子硫苷酶释放的硫代葡萄糖苷苷元自发重排的产物，但硫代乙腈和有机硫氰酸盐的形成既依赖于黑芥子硫苷酶，也依赖于特异性蛋白。许多含硫代葡萄糖苷的植物物种的水解产物谱表明存在特异性蛋白，但只有少数被鉴定并在生化上进行了表征。在这里，我们报告了来自野芝麻（十字花科）的硫氰酸盐形成蛋白（TFP）TaTFP 的 cDNA 克隆、异源表达和特性，该蛋白在所有植物器官中表达，并可在大肠杆菌中异源表达后以活性形式纯化。作为一个特殊特征，该蛋白在黑芥子硫苷酶催化水解烯丙基硫代葡萄糖苷时，既能促进丙烯硫氰酸盐的形成，也能促进相应的硫代乙腈的形成，烯丙基硫代葡萄糖苷是野芝麻的主要硫代葡萄糖苷。当用 TaTFP 水解时，所有其他测试的硫代葡萄糖苷都转化为其简单的腈。尽管 TaTFP 能够促进丙烯硫氰酸盐的形成，但它与已知的表硫代特异性蛋白（ESPs）的氨基酸序列相似性高于萝卜 TFP。然而，与拟南芥 ESP 不同，它在体外的活性并不严格依赖于向测定混合物中添加 Fe²⁺。TaTFP 以纯化形式提供，这使得未来的研究旨在阐明特异性蛋白特异性和机制的结构基础。此外，TaTFP 的鉴定表明，特异性蛋白的产物特异性不能基于氨基酸序列相似性来预测，并提出了关于特异性蛋白进化的有趣问题。
