Department of Biology, Norwegian University of Science and Technology-NTNU, Realfagbygget, Høgskoleringen 5, NO-7491 Trondheim, Norway.
Phytochemistry. 2012 Dec;84:7-17. doi: 10.1016/j.phytochem.2012.08.004. Epub 2012 Sep 3.
Glucosinolates are plant secondary metabolites that are part of a plant defence system against pathogens and pests, the myrosinase-glucosinolate system, in which glucosinolates get activated by enzymic degradation through thioglucoside glucohydrolases called myrosinases. Epithiospecifier protein (ESP) and nitrile-specifier proteins (NSPs) divert myrosinase-catalyzed hydrolysis of a given glucosinolate from the formation of isothiocyanate to that of epithionitrile and/or nitrile. As the biological activity of glucosinolate hydrolysis products varies considerably, a detailed characterization of these specifier proteins is of utmost importance to understand their biological role. Therefore, the Arabidopsis thaliana AtNSP1, AtNSP2 and AtNSP5 and a supposed ancestor protein AtNSP-like1 were expressed in Escherichia coli and the activity of the purified recombinant proteins was tested in vitro on three highly different glucosinolates and compared to that of purified AtESP. As previously reported, only AtESP showed epithiospecifier activity on 2-propenylglucosinolate. We further confirmed that purified AtNSP1, AtNSP2 and AtNSP5, but not the ancestor AtNSP-like1 protein, show nitrile-specifier activity on 2-propenylglucosinolate and benzylglucosinolate. We now show for the first time that in vitro AtNSP1, AtNSP2 and AtNSP5 are able to generate nitrile from indol-3-ylmethylglucosinolate. We also tested the effect of different Fe(II) ion concentrations on the nitrile-specifier activity of purified AtNSP1, AtNSP2 and AtNSP5 on 2-propenylglucosinolate and benzylglucosinolate. AtNSP-related nitrile production was highly dependent on the presence of Fe(II) ions in the reaction assay. In the absence of added Fe(II) ions nitriles were only detected when benzylglucosinolate was incubated with AtNSP1. While AtNSP1 also exhibited overall higher nitrile-specifier activity than AtNSP2 and AtNSP5 at a given Fe(II) ion concentration, the pattern of nitrile formation in relation to Fe(II) ion concentrations depended on the AtNSP and the glucosinolate substrate. The pH of the solution also affected the reaction outcome, with a higher proportion of nitrile being produced at the higher pH for AtNSP2 and AtNSP5.
硫代葡萄糖苷是植物次生代谢物，是植物防御系统的一部分，可抵御病原体和害虫，即通过称为黑芥子酶的硫葡糖苷葡糖水解酶的酶促降解来激活黑芥子酶-硫代葡萄糖苷系统。表硫苷酶蛋白 (ESP) 和腈指定蛋白 (NSP) 将黑芥子酶催化水解特定硫代葡萄糖苷的产物从异硫氰酸盐的形成转移到表硫腈和/或腈的形成。由于硫代葡萄糖苷水解产物的生物活性差异很大，因此对这些指定蛋白进行详细表征对于理解其生物学功能至关重要。因此，在大肠杆菌中表达了拟南芥 AtNSP1、AtNSP2 和 AtNSP5 以及假定的祖先蛋白 AtNSP-like1，并在体外使用三种高度不同的硫代葡萄糖苷对纯化的重组蛋白的活性进行了测试，并与纯化的 AtESP 进行了比较。如前所述，只有 AtESP 对丙烯基硫代葡萄糖苷显示出表硫苷特异性活性。我们进一步证实，纯化的 AtNSP1、AtNSP2 和 AtNSP5，但不是祖先 AtNSP-like1 蛋白，对丙烯基硫代葡萄糖苷和苄基硫代葡萄糖苷显示出腈指定活性。我们现在首次表明，体外 AtNSP1、AtNSP2 和 AtNSP5 能够从吲哚-3-基甲基硫代葡萄糖苷生成腈。我们还测试了不同 Fe(II) 离子浓度对纯化的 AtNSP1、AtNSP2 和 AtNSP5 在丙烯基硫代葡萄糖苷和苄基硫代葡萄糖苷上的腈指定活性的影响。在反应测定中存在 Fe(II) 离子时，与 AtNSP 相关的腈生成高度依赖于 Fe(II) 离子的存在。在没有添加 Fe(II) 离子的情况下，只有当苄基硫代葡萄糖苷与 AtNSP1 孵育时才检测到腈。虽然 AtNSP1 在给定的 Fe(II) 离子浓度下也表现出比 AtNSP2 和 AtNSP5 更高的腈指定活性，但腈的形成模式与 Fe(II) 离子浓度有关，这取决于 AtNSP 和硫代葡萄糖苷底物。溶液的 pH 也会影响反应结果，AtNSP2 和 AtNSP5 在较高 pH 时会产生更高比例的腈。
