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Characterization of plant glutamine synthetase S-nitrosation

机译：植物谷氨酰胺合成酶S-亚硝化的表征

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The identification of S-nitrosated substrates and their target cysteine residues is a crucial step to understand the signaling functions of nitric oxide (NO) inside the cells. Here, we show that the key nitrogen metabolic enzyme glutamine synthetase (GS) is a S-nitrosation target in Medicago truncatula and characterize the molecular determinants and the effects of this NO-induced modification on different GS isoenzymes. We found that all the four M. truncatula GS isoforms are S-nitrosated, but despite the high percentage of amino acid identity between the four proteins, S-nitrosation only affects the activity of the plastid-located enzymes, leading to inactivation. A biotin-switch/mass spectrometry approach revealed that cytosolic and plastid-located GSs share an S-nitrosation site at a conserved cysteine residue, but the plastidic enzymes contain additional S-nitrosation sites at non conserved cysteines, which are accountable for enzyme inactivation. By site-directed mutagenesis, we identified Cys369 as the regulatory S-nitrosation site relevant for the catalytic function of the plastid-located GS and an analysis of the structural environment of the SNO-targeted cysteines in cytosolic and plastid-located isoenzymes explains their differential regulation by S-nitrosation and elucidates the mechanistic by which S-nitrosation of Cys369 leads to enzyme inactivation. We also provide evidence that both the cytosolic and plastid-located GSs are endogenously S-nitrosated in leaves and root nodules of M. truncatula, supporting a physiological meaning for S-nitrosation. Taken together, these results provide new insights into the molecular details of the differential regulation of individual GS isoenzymes by NO-derived molecules and open new paths to explore the biological significance of the NO-mediated regulation of this essential metabolic enzyme.
机译：S-亚硝化底物及其靶半胱氨酸残基的鉴定是了解在细胞内部的一氧化氮（NO）的信号传导功能的关键步骤。这里，我们表明关键的氮代谢酶谷氨酰胺合成酶（GS）是Medicago Truncatula中的S-亚硝化靶标，表征分子决定因素和这种无诱导的修饰对不同GS同工酶的影响。我们发现，所有四个M.Truncatula GS同种型都是S-亚硝化，但尽管四种蛋白质之间的氨基酸同一性高，但S-亚硝化仅影响塑体定位的酶的活性，导致灭活。生物素 - 开关/质谱方法揭示了细胞溶质和塑体的GSS在保守的半胱氨酸残基中共享S-亚硝化位点，但塑性酶在非保守的半胱氨酸含有另外的S-亚硝化位点，这对酶失活负责。通过定点诱变，我们将Cys369确定为具有塑性化GS的催化功能的调节S-亚硝化位点，分析细胞溶质和塑性血浆中的SnO靶向半胱氨酸的结构环境解释了它们的差异通过S-亚硝化调节并阐明Cys369的S-亚硝化导致酶失活的机械。我们还提供了胞质溶胶和塑性体积的GSS在M. Truncatula的叶片和根结节中内源性S-亚硝化，支持S-亚硝化的生理学意义。总之，这些结果提供了No衍生的分子对单个GS同工酶的差异调节的分子细节的新见解，并开放新的途径，以探讨这种必需代谢酶的无介导调节的生物学意义。

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