Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanism
Molecular models and mutational analyses of plant specifier proteins suggest active site residues and reaction mechanism
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DOI：
10.1007/s11103-013-0126-0
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发表时间：
2014-01-01
影响因子：
5.1
通讯作者：
Wittstock, Ute
中科院分区：
文献类型：
--
作者：
Brandt, Wolfgang;Backenkoehler, Anita;Wittstock, Ute
作为芥子油苷-黑芥子酶系统的组成部分，特异性蛋白有助于植物中进化的化学防御的多样性，作为对食草动物和病原体的保护。芥子油苷是与其水解酶黑芥子酶分开储存在植物组织中的硫代葡萄糖苷。在组织破裂时，芥子油苷被黑芥子酶水解，产生不稳定的糖苷配基，这些糖苷配基重新排列形成防御性异硫氰酸酯。在特异性蛋白质的存在下，可以形成其他产物，即简单的腈、表硫腈和有机硫氰酸酯，而不是异硫氰酸酯，这取决于芥子油苷侧链结构和特异性蛋白质的类型。特异性蛋白的生化作用在很大程度上还没有得到解决。我们已经使用了两个硫氰酸盐形成蛋白和一个epithiospecifier蛋白与不同的底物/产品的特异性开发的分子模型，结合突变分析，使我们能够提出一个活性位点和对接安排与硫代葡萄糖苷配基，可以解释一些特异性的差异。此外，量子力学计算支持硫氰酸苄酯形成的反应机制，包括所涉及的TFP的催化作用。这些结果可能作为进一步的理论和实验研究的基础上，硫代葡萄糖甙的分解机制，也将有助于更好地了解从祖先蛋白质的进化与功能以外的硫代葡萄糖甙代谢。
As components of the glucosinolate-myrosinase system, specifier proteins contribute to the diversity of chemical defenses that have evolved in plants of the Brassicales order as a protection against herbivores and pathogens. Glucosinolates are thioglucosides that are stored separately from their hydrolytic enzymes, myrosinases, in plant tissue. Upon tissue disruption, glucosinolates are hydrolyzed by myrosinases yielding instable aglucones that rearrange to form defensive isothiocyanates. In the presence of specifier proteins, other products, namely simple nitriles, epithionitriles and organic thiocyanates, can be formed instead of isothiocyanates depending on the glucosinolate side chain structure and the type of specifier protein. The biochemical role of specifier proteins is largely unresolved. We have used two thiocyanate-forming proteins and one epithiospecifier protein with different substrate/product specificities to develop molecular models that, in conjunction with mutational analyses, allow us to propose an active site and docking arrangements with glucosinolate aglucones that may explain some of the differences in specifier protein specificities. Furthermore, quantum-mechanical calculations support a reaction mechanism for benzylthiocyanate formation including a catalytic role of the TFP involved. These results may serve as a basis for further theoretical and experimental investigations of the mechanisms of glucosinolate breakdown that will also help to better understand the evolution of specifier proteins from ancestral proteins with functions outside glucosinolate metabolism.