Identification and characterization cytosolic nucleoside monophosphate phosphatases of plants

植物胞浆核苷单磷酸磷酸酶的鉴定和表征

• 批准号: 462603278
• 负责人: Professor Dr. Claus-Peter Witte
• 金额: --
• 依托单位: Department of Agricultural Biotechnology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/462603278?language=en
• 关键词: Identification; characterization; cytosolic; nucleoside; monophosphate

Plant genomes code for a large number of phosphatases, but the physiological function of only a few is known. The difficulties in determining the in vivo function of phosphatases result among others from the fact that the substrate specificities in vitro are often low and that the enzymes belong to (functionally redundant) protein families.In plant nucleotide metabolism, the phosphatases that convert mononucleotides into the corresponding nucleosides in vivo are also still unknown. However, it is clear that such phosphatases must exist, because plants are able to completely degrade nucleotides.In this project, cytosolic mononucleotide phosphatases for both purine and pyrimidine nucleotides will be identified and biochemically characterized, and their in vivo position and function in metabolism will be investigated.Pyrimidine mononucleotide phosphatases from yeast were chosen as the starting point because they have a demonstrated role in nucleotide metabolism in vivo and because homologous enzymes occur in plants. The subcellular localization and substrate specificity of five homologous phosphatases from Arabidopsis thaliana were investigated. All enzymes were present in the cytosol. One enzyme dephosphorylated exclusively the purine nucleotide xanthosine monophosphate (XMP) with high catalytic efficiency. The existence of a plant XMP phosphatase (XMPP) has already been postulated by us on the basis of metabolite studies on Arabidopsis mutants. The biochemistry of the Arabidopsis XMPP will be comprehensively studied. The position and function of XMPP in metabolism will be investigated using metabolite analyses of mutants. The XMPP of common bean (Phaseolus vulgaris) will also be characterized and the gene will be mutated in transgenic roots / nodules using gene scissors. Metabolite analyses on xmpp nodules will clarify whether XMPP plays an important role in the generation of intermediates in purine catabolism (the ureides), which are used to export fixed nitrogen from the nodules. For this purpose, also the expression domain of the XMPP gene in the nodule will be examined.The remaining four phosphatases and a further phosphatase with homology to a human nucleotidase showed a high specificity for pyrimidine mononucleotides. These enzymes will be biochemically characterized in detail. Metabolite analyses in mutants and possibly multiple mutants will be performed in order to elucidate the physiological functions of these enzymes and to investigate their role in vivo, for example upon increased nucleotide degradation in the dark. Promoter-reporter studies will be performed to elucidate in which cell types and tissues the phosphatase genes are active. By integrating these data, a first model for the role of pyrimidine mononucleotide-specific phosphatases in nucleotide metabolism will be developed.

植物基因组编码大量的磷酸酶，但只有少数的生理功能是已知的。确定磷酸酶在体内功能的困难是由于底物在体外的特异性通常很低，而且酶属于（功能冗余的）蛋白质家族。在植物核苷酸代谢中，体内将单核苷酸转化为相应核苷的磷酸酶也尚不清楚。然而，很明显这种磷酸酶一定存在，因为植物能够完全降解核苷酸。本项目将对嘌呤和嘧啶核苷酸的胞质单核苷酸磷酸酶进行鉴定和生化表征，并研究其在体内的位置和代谢功能。之所以选择酵母菌中的嘧啶单核苷酸磷酸酶作为起点，是因为它们在体内核苷酸代谢中具有明确的作用，而且同源酶也存在于植物中。研究了拟南芥5种同源磷酸酶的亚细胞定位和底物特异性。所有酶均存在于细胞质中。一种酶具有高催化效率，可以完全去磷酸化嘌呤核苷酸单磷酸黄嘌呤（XMP）。植物XMP磷酸酶（XMPP）的存在是我们在拟南芥突变体代谢产物研究的基础上提出的假设。对拟南芥XMPP的生化特性进行全面研究。XMPP在代谢中的位置和功能将通过突变体的代谢物分析来研究。对菜豆（Phaseolus vulgaris）的XMPP进行鉴定，并利用基因剪刀在转基因根/根瘤中进行基因突变。对xmpp结核的代谢物分析将阐明xmpp是否在嘌呤分解代谢的中间体（ureides）的生成中起重要作用，这些中间体用于从结核中输出固定氮。为此，还将检查XMPP基因在结节中的表达域。其余四种磷酸酶和另一种与人类核苷酸酶同源的磷酸酶对嘧啶单核苷酸具有高特异性。这些酶将进行详细的生物化学表征。为了阐明这些酶的生理功能，并研究它们在体内的作用，例如在黑暗中核苷酸降解增加的情况下，将对突变体和可能的多突变体进行代谢物分析。启动子报告子研究将用于阐明哪些细胞类型和组织中磷酸酶基因是活跃的。通过整合这些数据，将开发出嘧啶单核苷酸特异性磷酸酶在核苷酸代谢中的作用的第一个模型。