Biochemical and physiological characterization of nucleoside kinases from Arabidopsis thaliana involved in the regulation of nucleotide biosynthesis

回收反应用于回收核苷，用于核苷酸和核酸的生物合成。腺苷、尿苷/胞苷和假尿苷的核苷激酶在植物中是已知的。我们最近描述了一种肌苷激酶，但在植物或其他真核生物中还不知道对应的鸟苷激酶，尽管放射性标记实验和活性测量表明它们肯定存在。我们已经鉴定了未知功能的激酶，以寻找新的核苷激酶。我们发现了一种胞质肌苷激酶(PNK1)，它可以磷酸化肌苷和尿苷，并参与体内肌苷的回收。PNK1突变导致嘌呤核苷酸分解代谢通量增加，在尿苷降解受损的情况下，尿苷和UTP过度积累，生长抑制。这些数据表明，PNK1参与了嘌呤核苷酸生物合成的反馈调节，也可能参与了嘧啶核苷酸生物合成的反馈调节。此外，还鉴定了另一种核苷激酶(工作名称：K6)。除了肌苷，它还可以使鸟苷磷酸化。K6基因突变导致幼苗中肌苷和肌苷一磷酸含量增加，并导致莲座状生长抑制。K6可能也参与了嘌呤生物合成的调节。这项研究的中心目标是进一步阐明PNK1和K6的生理功能。对于PNK1，已经为此做了大量的工作，例如创造了双突变体和互补系。对于K6来说，这项工作仍然需要完成。通过对遗传变异的代谢物分析和15N标记实验，将研究这两种酶在核苷酸代谢反馈控制中的功能。核苷酸的生物合成也受到TOR激酶的更高水平的控制，TOR激酶本身可以受到核苷酸的调节，尽管细节仍不清楚。监管层面(TOR监管和反馈监管)可能的相互作用将在合作中进行调查。在另一项合作中，结合底物的激酶(S)的蛋白质结构也将被阐明，以了解底物专一性的分子细节。可选的是，进一步寻找核苷激酶的工作也应继续进行。具体地说，如果在项目期间有足够的资源可用，可以研究两种候选酶。所有要研究的激酶在植物中都是高度保守的，有些显然参与了核苷酸代谢的调节。因此，这项研究项目开辟了新的科学基础，因为植物核苷酸代谢的调节方面在很大程度上仍然未知。

Salvage reactions are used to recycle nucleosides for the biosynthesis of nucleotides and nucleic acids. Nucleoside kinases for adenosine, uridine/cytidine and pseudouridine are known in plants. An inosine kinase was recently described by us, but corresponding kinases for guanosine are not yet known in plants or other eukaryotes, although radioactive labeling experiments and activity measurements demonstrate that they must exist. We have characterized kinases of unknown function to find new nucleoside kinases. We identified a plastidic inosine kinase (PNK1) that can phosphorylate inosine and uridine and is involved in the salvage of inosine in vivo. Mutation of PNK1 results in increased flux to purine nucleotide catabolism and, in the context of impaired uridine degradation, overaccumulation of uridine and UTP and growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. Furthermore, another nucleoside kinase (working name: K6) has been identified. In addition to inosine, this can also phosphorylate guanosine. Mutation of K6 leads to increased inosine and inosine monophosphate content in seedlings and to rosette growth depression. K6 may also contribute to the regulation of purine biosynthesis. The central goal of the proposed research is to further elucidate the physiological functions of PNK1 and K6. For PNK1, extensive work has already been done for this purpose, such as the creation of double mutants and complementation lines. This work would still need to be done for K6. A function in feedback control of nucleotide metabolism will be investigated for both kinases by metabolite analyses of genetic variants and 15N labeling experiments. Nucleotide biosynthesis is also controlled in a higher level by the TOR kinase, which itself can be regulated by nucleotides, although the details are still unknown. Possible interactions of the regulatory levels (TOR regulation and feedback regulation) will be investigated in a collaboration. In another collaboration, the protein structures of the kinases with bound substrate(s) will also be elucidated to understand the molecular details of the substrate specificity of the kinases. Optionally, the search for further nucleoside kinases should also be continued. Specifically, two candidate enzymes could be studied if sufficient resources are available during the project. All kinases to be studied here are highly conserved in plants, and some are apparently involved in the regulation of nucleotide metabolism. Thus, this research project breaks new scientific ground, because regulatory aspects of plant nucleotide metabolism are still largely unknown.