Asparagine metabolism and function in nitrogen signaling

氨基酸天冬酰胺是氮的主要形式，通常运输到高等植物的汇组织，包括根、花和发育中的种子，由于其高氮碳比。生殖组织中天冬酰胺的局部生物合成也可以为谷物储存储备积累提供先决条件。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，有机酸的水平不足以同化氮。天冬酰胺是光呼吸的重要中间体，提供氮的输入来平衡其他途径中使用的丝氨酸或甘氨酸的输出。本提案的目的是实现对天冬酰胺代谢的详细理解，以及天冬酰胺作为有机氮代谢信号的功能和感知。报告筛选先前确定的天冬酰胺反应基因将有助于确定参与其调节的因素。基因调控网络也将通过计算机启动子分析、同源调控物鉴定和诱导基因谱分析进行研究。对天冬酰胺代谢的光呼吸途径进行生物化学研究。酶- omega-酰胺酶参与天冬酰胺转氨化反应产物α -酮琥珀酸酯及其还原形式α -羟基琥珀酸酯的分解。它与一种酶具有高度的序列同一性，这种酶催化由转氨化引起的抗氧化剂谷胱甘肽的损坏形式的分解。这些酶的底物特异性的结构决定因素将通过结合蛋白质晶体学和定点诱变的方法来确定。将确定负责还原α -酮琥珀酸盐的脱氢酶。将采用两种可供选择的方法。首先，生物信息学方法将检测模式植物拟南芥基因组中天冬酰胺代谢和光呼吸基因与脱氢酶的共表达。候选基因将通过测定相应重组酶的体外催化活性来表征。第二种方法将涉及从大豆叶片中纯化酶，并通过位点特异性蛋白酶消化，质谱分析和蛋白质组数据库搜索来鉴定纯化蛋白。候选基因将通过分析拟南芥在外源非标记和稳定标记的天冬酰胺处理下相应敲除突变体的代谢物来验证。研究结果将有助于我们对高等植物中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.