London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada; Department of Biology, University of Western Ontario, 1151 Richmond Street, London, Ontario, N6A 5B7, Canada.
London Research and Development Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada.
Plant Sci. 2021 Nov;312:111033. doi: 10.1016/j.plantsci.2021.111033. Epub 2021 Aug 25.
The glutamine amidotransferase gene GAT1_2.1 is a marker of N status in Arabidopsis root, linked to a shoot branching phenotype. The protein has an N-terminal glutamine amidotransferase domain and a C-terminal extension with no recognizable protein domain. A purified, recombinant version of the glutamine amidotransferase domain was catalytically active as a glutaminase, with apparent K value of 0.66 mM and V value of 2.6 μkatal per mg. This form complemented an E. coli glutaminase mutant, ΔYneH. Spiking of root metabolite extracts with either the N-terminal or full length form purified from transformed tobacco leaves led to reciprocal changes in glutamine and ammonia concentration. No product derived from amido-N-labeled glutamine was identified. Visualization of GAT1_2.1-YPF transiently expressed in tobacco leaves confirmed its mitochondrial localization. gat1_2.1 exhibited reduced growth as compared with wild-type seedlings on media with glutamine as sole nitrogen source. Results of targeted metabolite profiling pointed to a possible activation of the GABA shunt in the mutant following glutamine treatments, with reduced levels of glutamic acid, 2-oxoglutarate and γ-aminobutyric acid and increased levels of succinic acid. GAT1_2.1 may act as a glutaminase, in concert with Glutamate Dehydrogenase 2, to hydrolyze glutamine and channel 2-oxoglutarate to the TCA cycle under high nitrogen conditions.
谷氨酰胺酰胺基转移酶基因 GAT1_2.1 是拟南芥根中氮状态的标志物，与分枝表型有关。该蛋白具有 N 端谷氨酰胺酰胺基转移酶结构域和 C 端延伸结构域，后者没有可识别的蛋白结构域。纯化的、重组形式的谷氨酰胺酰胺基转移酶结构域具有谷氨酰胺酶的催化活性，表观 K 值为 0.66 mM，V 值为 2.6 μkatal/mg。这种形式可以补充大肠杆菌谷氨酰胺酶突变体 ΔYneH。向根代谢物提取物中添加从转化的烟草叶片中纯化的 N 端或全长形式，导致谷氨酰胺和氨浓度发生相互变化。未鉴定出源自酰胺-N 标记的谷氨酰胺的产物。在烟草叶片中转瞬表达的 GAT1_2.1-YPF 的可视化证实了其在线粒体中的定位。与野生型幼苗相比， gat1_2.1 在以谷氨酰胺为唯一氮源的培养基上的生长速度较慢。靶向代谢物分析的结果表明，突变体在谷氨酰胺处理后 GABA 支路可能被激活，谷氨酸、2-酮戊二酸和γ-氨基丁酸水平降低，琥珀酸水平升高。GAT1_2.1 可能与谷氨酸脱氢酶 2 一起作为谷氨酰胺酶，在高氮条件下水解谷氨酰胺并将 2-酮戊二酸转运到 TCA 循环。
