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

• Applicant: Professor Dr. Claus-Peter Witte
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 528245572

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.

• DFG Programme: Research Grants
• International Connection: South Korea, USA
• Cooperation Partners: Professor Dr. Jake Brunkard; Professor Dr. Sangkee Rhee