Identification and characterization cytosolic nucleoside monophosphate phosphatases of plants

植物基因组编码了大量的磷酸酶，但只有几个的生理功能是已知的。磷酸酶在体内的功能难以确定，原因之一是体外底物特异性低，而且这些酶属于(功能冗余的)蛋白质家族。在植物核苷酸代谢中，在体内将单核苷酸转化为相应核苷的磷酸酶也是未知的。然而，很明显，这样的磷酸酶是存在的，因为植物能够完全降解核苷酸。在这个项目中，将鉴定和生化鉴定胞浆中的嘌呤和嘧啶核苷酸的单核苷酸磷酸酶，并研究它们在体内的位置和在代谢中的功能。选择酵母中的嘧啶单核苷酸磷酸酶作为起点，因为它们在体内的核苷酸代谢中具有证明的作用，并且因为同源酶在植物中存在。研究了拟南芥5种同源磷酸酶的亚细胞定位和底物专一性。所有的酶都存在于胞浆中。一种酶以高催化效率仅对嘌呤核苷酸黄嘌呤一磷酸(XMP)进行脱磷。植物XMP磷酸酶(XMPP)的存在已被我们根据对拟南芥突变体代谢物的研究而推测。将对拟南芥XMPP的生物化学进行全面研究。XMPP在代谢中的地位和作用将通过对突变体的代谢物分析来研究。还将研究菜豆的XMPP，并利用基因剪刀在转基因根/瘤中对该基因进行突变。对XMPP结核的代谢物分析将澄清XMPP是否在产生嘌呤分解代谢中间产物(脲)方面发挥重要作用，这些中间产物用于从结核输出固定氮。为此目的，还将检查XMPP基因在结节中的表达结构域。其余四种磷酸酶和另一种与人核苷酸酶同源的磷酸酶对嘧啶单核苷酸具有高度的特异性。将对这些酶进行详细的生化表征。将对突变体和可能的多个突变体进行代谢物分析，以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。将进行启动子-报告者研究，以阐明在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。

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.