The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism
The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism
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DOI：
10.1007/s11103-015-0312-3
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发表时间：
2015-05-01
影响因子：
5.1
通讯作者：
Binder, Stefan
中科院分区：
文献类型：
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作者：
Laechler, Kurt;Imhof, Janet;Binder, Stefan
拟南芥（Arabidopsis thaliana）具有六种支链氨基转移酶（BCAT 1 -6）。以前的研究表明，该蛋白质家族的一些成员参与支链氨基酸的生物合成和/或Met链延伸途径，这是Met衍生的芥子油苷生物合成的初始步骤。我们现在分析了支链氨基转移酶6（BCAT 6）。体内GFP标记实验强烈表明这种酶定位于细胞质。用重组酶进行的底物特异性测定显示，BCAT 6不仅能转氨瓦尔、Leu和Ile以及相应的2-氧代酸，而且还能转氨Met及其同源酮酸4-甲基-2-氧代丁酸酯。我们建立了涉及BCAT 6的单突变体（bcat 6 -1）、双突变体（bcat 4 -2/bcat 6 -1）和三突变体（bcat 3 -1/bcat 4 -2/bcat 6 -1），后者表现出清晰的宏观表型，具有较小的植物和异常的叶片形态。这些突变体的代谢物分析表明，BCAT 6可以有助于Met链延长，缺乏BCAT 3、4和6的三重突变体系显示出Met衍生的硫代葡萄糖苷种类在植物叶子和种子中分别显著减少至野生型水平的32%和14%。这种芥子油苷水平的下降伴随着游离Met的46倍增加，证明了三种支链氨基转移酶在将Met转化为其2-含氧酸以延长芥子油苷链中的重要作用。此外，我们确定了5 '-脱氧-5'-甲硫基腺苷（Met再循环途径的中间体）的相对量。在不存在胞质BCAT 4和BCAT 6的情况下，这种代谢物积累到相对高的量，表明胞质Met补救也有助于芥子油苷的生物合成。
Arabidopsis thaliana possesses six branched-chain aminotransferases (BCAT1-6). Previous studies revealed that some members of this protein family are involved in the biosynthesis of branched-chain amino acids and/or in the Met chain elongation pathway, the initial steps towards the biosynthesis of Met-derived glucosinolates. We now analyzed branched-chain aminotransferase 6 (BCAT6). In vivo GFP-tagging experiments strongly suggest this enzyme to be localized to the cytosol. Substrate specificity assays performed with recombinant enzyme revealed that BCAT6 transaminates Val, Leu and Ile as well as the corresponding 2-oxo acids but also transaminates Met and its cognate ketoacid 4-methyl-2-oxobutanoate. We established single (bcat6-1), double (bcat4-2/bcat6-1) and triple (bcat3-1/bcat4-2/bcat6-1) mutants involving BCAT6 with the latter exhibiting a clear macroscopic phenotype with smaller plants and abnormal leaf morphology. Metabolite profiling of these mutants demonstrated that BCAT6 can contribute to Met chain elongation with the triple mutant line lacking BCAT3, 4 and 6 showing a dramatic reduction of Met-derived glucosinolate species down to 32 and 14 % of wild-type levels in plant foliage and seeds, respectively. This drop in glucosinolate levels is accompanied by a 46-fold increase of free Met, demonstrating the important role of the three branched-chain aminotransferases in converting Met to its 2-oxo acid for glucosinolate chain elongation. In addition, we determined the relative amounts of 5'-deoxy-5'-methylthioadenosine, an intermediate of the Met recycling pathway. This metabolite accumulated to relative high amounts in the absence of the cytosolic BCAT4 and BCAT6, suggesting that cytosolic Met salvage also contributes to the biosynthesis of glucosinolates.