Asparagine metabolism and function in nitrogen signaling

氨基酸天冬酰胺是一种主要的氮素形式，由于其高的氮碳比，通常被运输到高等植物的下沉组织中，包括根、花和发育中的种子。生殖组织中天冬酰胺的局部生物合成也为籽粒储藏积累提供了条件。在叶片中，天冬酰胺在夜间短暂积累，在没有光合作用的情况下，有机酸水平不足以进行氮同化。天冬酰胺是光呼吸的重要中间体，提供氮的输入以平衡用于其他途径的丝氨酸或甘氨酸的输出。这项建议的目的是实现对天冬酰胺代谢的详细了解，以及天冬酰胺作为高等植物有机氮代谢产物信号的功能和认知。记者筛选之前发现的天冬酰胺反应基因将有助于识别参与其调控的因素。基因调控网络还将通过电子启动子分析、同源调节子鉴定和诱导基因图谱分析进行研究。天冬酰胺代谢的光呼吸途径的生物化学将被研究。酶omega-amidase参与了天冬酰胺转氨化反应产物α-酮基琥珀酸酯及其还原形式α-羟基丁二酸酯的分解。它与一种酶有高度的序列同源性，该酶催化分解转氨基作用产生的一种受损形式的抗氧化剂谷胱甘肽。这些酶中底物专一性的结构决定因素将通过结合蛋白质结晶学和定点突变的方法来鉴定。负责还原α-酮基丁二酸酯的脱氢酶将被确定。将使用两种替代方法。首先，生物信息学方法将检测天冬酰胺代谢和光呼吸基因与脱氢酶在模式植物拟南芥基因组中的共表达。候选基因将通过在体外测定相应重组酶的催化活性来表征。第二种方法将包括从大豆叶片中提纯酶，并通过定点特定的蛋白酶、质谱分析和蛋白质组数据库搜索来鉴定纯化的蛋白质。候选基因将通过分析相应的突变体中的代谢物来验证，该突变体对外源非标记和稳定标记的天冬酰胺的处理做出了反应。这一研究结果将加深我们对高等植物天冬酰胺代谢和信号转导的认识。

The amino acid asparagine is a major form of nitrogen commonly transported to sink tissues of higher plants, including roots, flowers and developing seeds, due to its high nitrogen to carbon ratio. Local biosynthesis of asparagine in reproductive tissues can also pre-condition storage reserve accumulation in grain. In leaf, asparagine is accumulated transiently at night when, in the absence of photosynthesis, levels of organic acids are insufficient for nitrogen assimilation. Asparagine is an important intermediate in photorespiration, providing an input of nitrogen to balance an output of serine or glycine used in other pathways. The objectives of this proposal are to achieve a detailed understanding of asparagine metabolism and of the function and perception of asparagine as a metabolite signal of organic nitrogen in higher plants. Reporter screens with previously identified asparagine-responsive genes will help identify factors involved in their regulation. Gene regulatory networks will also be investigated by in silico promoter analysis, identification of cognate regulators and inducible gene profiling assays. The biochemistry of the photorespiratory pathway of asparagine metabolism will be investigated. The enzyme omega-amidase is involved in the breakdown of the reaction product of asparagine transamination, alpha-ketosuccinamate and of its reduced form, alpha-hydroxysuccinamate. It shares a high degree of sequence identity with an enzyme catalyzing the breakdown of a damaged form of the antioxidant glutathione, arising from transamination. The structural determinants of substrate specificity in these enzymes will be identified by an approach combining protein crystallography and site-directed mutagenesis. The dehydrogenase enzyme responsible for the reduction of alpha-ketosuccinamate will be identified. Two alternative approaches will be used. First, a bioinformatic approach will examine co-expression of asparagine metabolic and photorespiratory genes with dehydrogenases in the genome of the model plant Arabidopsis thaliana. Candidate genes will be characterized by determining the catalytic activity of the corresponding recombinant enzyme in vitro. The second approach will involve purification of the enzyme from soybean leaves, and identification of the purified protein by digestion with a site-specific protease, mass spectrometry and search against a proteome database. Candidate genes will be validated by profiling metabolites in the corresponding knock-out mutant of A. thaliana in response to treatment with exogenous non-labeled and stably labeled asparagine. The research results will improve our understanding of asparagine metabolism and signaling in higher plants.