Kissen Ralph, Bones Atle M
Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway.
J Biol Chem. 2009 May 1;284(18):12057-70. doi: 10.1074/jbc.M807500200. Epub 2009 Feb 18.
Glucosinolates are plant secondary metabolites present in Brassicaceae plants such as the model plant Arabidopsis thaliana. Intact glucosinolates are believed to be biologically inactive, whereas degradation products after hydrolysis have multiple roles in growth regulation and defense. The degradation of glucosinolates is catalyzed by thioglucosidases called myrosinases and leads by default to the formation of isothiocyanates. The interaction of a protein called epithiospecifier protein (ESP) with myrosinase diverts the reaction toward the production of epithionitriles or nitriles depending on the glucosinolate structure. Here we report the identification of a new group of nitrile-specifier proteins (AtNSPs) in A. thaliana able to generate nitriles in conjunction with myrosinase and a more detailed characterization of one member (AtNSP2). Recombinant AtNSP2 expressed in Escherichia coli was used to test its impact on the outcome of glucosinolate hydrolysis using a gas chromatography-mass spectrometry approach. AtNSP proteins share 30-45% sequence homology with A. thaliana ESP. Although AtESP and AtNSP proteins can switch myrosinase-catalyzed degradation of 2-propenylglucosinolate from isothiocyanate to nitrile, only AtESP generates the corresponding epithionitrile. Using the aromatic benzylglucosinolate, recombinant AtNSP2 is also able to direct product formation to the nitrile. Analysis of glucosinolate hydrolysis profiles of transgenic A. thaliana plants overexpressing AtNSP2 confirms its nitrile-specifier activity in planta. In silico expression analysis reveals distinctive expression patterns of AtNSPs, which supports a biological role for these proteins. In conclusion, we show that AtNSPs belonging to a new family of A. thaliana proteins structurally related to AtESP divert product formation from myrosinase-catalyzed glucosinolate hydrolysis and, thereby, likely affect the biological consequences of glucosinolate degradation. We discuss similarities and properties of AtNSPs and related proteins and the biological implications.
硫代葡萄糖苷是十字花科植物（如模式植物拟南芥）中的植物次生代谢产物。完整的硫代葡萄糖苷被认为是无生物活性的，而水解后的降解产物在生长调节和防御中具有多种作用。硫代葡萄糖苷的降解由称为黑芥子酶的硫代葡萄糖苷酶催化，默认情况下会导致异硫氰酸酯的形成。一种称为表硫特异蛋白（ESP）的蛋白质与黑芥子酶的相互作用会根据硫代葡萄糖苷的结构将反应导向环硫腈或腈的生成。在这里，我们报告了在拟南芥中鉴定出一组新的腈特异蛋白（AtNSPs），它们能够与黑芥子酶一起生成腈，并对其中一个成员（AtNSP2）进行了更详细的表征。使用气相色谱 - 质谱分析法，检测了在大肠杆菌中表达的重组AtNSP2对硫代葡萄糖苷水解结果的影响。AtNSP蛋白与拟南芥ESP具有30 - 45%的序列同源性。虽然AtESP和AtNSP蛋白都能将黑芥子酶催化的2 - 丙烯基硫代葡萄糖苷降解从异硫氰酸酯转换为腈，但只有AtESP能生成相应的环硫腈。使用芳香族苄基硫代葡萄糖苷时，重组AtNSP2也能够将产物形成导向腈。对过表达AtNSP2的转基因拟南芥植物硫代葡萄糖苷水解谱的分析证实了其在植物中的腈特异活性。计算机模拟表达分析揭示了AtNSPs独特的表达模式，这支持了这些蛋白质的生物学作用。总之，我们表明，属于拟南芥中与AtESP结构相关的新蛋白家族的AtNSPs改变了黑芥子酶催化的硫代葡萄糖苷水解的产物形成，从而可能影响硫代葡萄糖苷降解的生物学后果。我们讨论了AtNSPs和相关蛋白的相似性、特性以及生物学意义。
