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

拟南芥核苷激酶参与核苷酸生物合成调节的生化和生理学特征

• 批准号: 528245572
• 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/528245572?language=en
• 关键词: Biochemical; physiological; characterization; nucleoside; kinases

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.

回收反应用于回收核苷以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相应的鸟苷激酶，尽管放射性标记实验和活性测量表明它们一定存在。我们已经确定了未知功能的激酶的特征，以发现新的核苷激酶。我们鉴定了一种质体肌苷激酶（PNK 1），它可以磷酸化肌苷和尿苷，并参与体内肌苷的挽救。PNK 1的突变导致嘌呤核苷酸催化剂的通量增加，并且在尿苷降解受损的情况下，尿苷和UTP的过度积累和生长抑制。这些数据表明，PNK 1参与嘌呤核苷酸生物合成的反馈调节，也可能参与嘧啶核苷酸生物合成。此外，还鉴定了另一种核苷激酶（工作名称：K6）。除了肌苷，它还可以磷酸化鸟苷。K6突变导致幼苗中肌苷和肌苷一磷酸含量增加，莲座丛生长抑制。K6也可能有助于嘌呤生物合成的调节。这项研究的中心目标是进一步阐明PNK 1和K6的生理功能。对于PNK 1，人们已经为此做了大量工作，例如创建双突变体和互补系。这一点在K6中仍然需要做。通过遗传变异的代谢物分析和15 N标记实验，将研究这两种激酶在核苷酸代谢反馈控制中的功能。核苷酸生物合成也在更高水平上由TOR激酶控制，TOR激酶本身可以由核苷酸调节，尽管细节仍然未知。监管层面（职权范围监管和反馈监管）的可能相互作用将在合作中进行调查。在另一项合作中，还将阐明激酶与结合底物的蛋白质结构，以了解激酶底物特异性的分子细节。任选地，还应继续寻找其他核苷激酶。具体而言，如果在项目期间有足够的资源，可以研究两种候选酶。这里要研究的所有激酶在植物中都是高度保守的，有些显然参与核苷酸代谢的调节。因此，这项研究项目开辟了新的科学领域，因为植物核苷酸代谢的调控方面在很大程度上仍然是未知的。