TY - BOOK

T1 - Characterization of three uridine monophosphate kinases from arabidopsis thaliana

AU - Rinne, Jannis

PY - 2024/4/2

Y1 - 2024/4/2

N2 - De novo synthesis of pyrimidine nucleotides is an essential pathway of plant primary metabolism that ends with UMP. This UMP serves as a precursor for the synthesis of all other pyrimidine nucleotides in plants. For this purpose, UMP is first phosphorylated by UMP KINASEs (UMKs), of which two families exist in plants: Eubacterial and AMP KINASE (AMK)-like UMKs. It is suggested that most UMK activity in vivo is catalyzed by AMK-like UMKs, of which Arabidopsis thaliana possesses three isoforms called UMK1, UMK2 and UMK3. Although the phosphorylation of UMP is of high importance for plant metabolism, the family has only been sparsely described so far. The aim of this thesis was to characterize the family of AMK-like UMKs of Arabidopsis and elucidate the specific roles of the three UMKs in pyrimidine nucleotide metabolism. While not all vascular plants possess a UMK1 gene, UMK2 and UMK3 are evolutionary conserved throughout the plant kingdom. Biochemical characterization showed that all three UMKs can phosphorylate UMP, CMP and dCMP. UMK3 stood out in this analysis as it possessed the highest catalytic efficiencies for UMP and CMP. Studies on subcellular localization showed that UMK1 and UMK3 are cytosolic enzymes, while UMK2 resides in mitochondria. The analysis of CRISPR/Cas9 mutant lines revealed a central function of UMK3 in pyrimidine nucleotide metabolism. A weak mutant of UMK3, encoding a partially defective enzyme, showed growth deficiencies and an altered pyrimidine nucleotide content, whereas null mutant alleles of UMK3 could not be inherited. While UMK3 is essential during the reproductive phase and for embryo development, mutants of UMK1 and UMK2 were phenotypically normal. In the background of the weak UMK3 mutant, in vivo functions of the other UMKs were unmasked. UMK2 mainly operates as dCMP kinase, and during germination, UMK2 activity is required for normal mitochondrial DNA replication. Also UMK3 was shown to be important for mitochondrial DNA replication during germination, indicating that mitochondria are capable of importing pyrimidine nucleotides from the cytosol. The data also suggest that mitochondria are able to export pyrimidine nucleotides, implying the existence of a mitochondrial pyrimidine nucleotide transporter. The role of UMK1 was less clear. UMK1 appears to be mainly involved in the phosphorylation of CMP, although this function was only observed in the background of the weak UMK3 mutant. It is possible that UMK1 is more important when the activity of UMK3 is downregulated. That such regulation may exist is indicated by the inhibition of UMK3 at high substrate concentrations. In such a situation, the activity of UMK1 could be important for the recycling of NMPs from DNA/RNA breakdown or nucleoside salvaging. Although the Arabidopsis AMK-like UMKs are all ubiquitously expressed and have similar enzymatic activities, this work shows that they are not redundant or at best partially redundant, but fulfill specific biological functions.

AB - De novo synthesis of pyrimidine nucleotides is an essential pathway of plant primary metabolism that ends with UMP. This UMP serves as a precursor for the synthesis of all other pyrimidine nucleotides in plants. For this purpose, UMP is first phosphorylated by UMP KINASEs (UMKs), of which two families exist in plants: Eubacterial and AMP KINASE (AMK)-like UMKs. It is suggested that most UMK activity in vivo is catalyzed by AMK-like UMKs, of which Arabidopsis thaliana possesses three isoforms called UMK1, UMK2 and UMK3. Although the phosphorylation of UMP is of high importance for plant metabolism, the family has only been sparsely described so far. The aim of this thesis was to characterize the family of AMK-like UMKs of Arabidopsis and elucidate the specific roles of the three UMKs in pyrimidine nucleotide metabolism. While not all vascular plants possess a UMK1 gene, UMK2 and UMK3 are evolutionary conserved throughout the plant kingdom. Biochemical characterization showed that all three UMKs can phosphorylate UMP, CMP and dCMP. UMK3 stood out in this analysis as it possessed the highest catalytic efficiencies for UMP and CMP. Studies on subcellular localization showed that UMK1 and UMK3 are cytosolic enzymes, while UMK2 resides in mitochondria. The analysis of CRISPR/Cas9 mutant lines revealed a central function of UMK3 in pyrimidine nucleotide metabolism. A weak mutant of UMK3, encoding a partially defective enzyme, showed growth deficiencies and an altered pyrimidine nucleotide content, whereas null mutant alleles of UMK3 could not be inherited. While UMK3 is essential during the reproductive phase and for embryo development, mutants of UMK1 and UMK2 were phenotypically normal. In the background of the weak UMK3 mutant, in vivo functions of the other UMKs were unmasked. UMK2 mainly operates as dCMP kinase, and during germination, UMK2 activity is required for normal mitochondrial DNA replication. Also UMK3 was shown to be important for mitochondrial DNA replication during germination, indicating that mitochondria are capable of importing pyrimidine nucleotides from the cytosol. The data also suggest that mitochondria are able to export pyrimidine nucleotides, implying the existence of a mitochondrial pyrimidine nucleotide transporter. The role of UMK1 was less clear. UMK1 appears to be mainly involved in the phosphorylation of CMP, although this function was only observed in the background of the weak UMK3 mutant. It is possible that UMK1 is more important when the activity of UMK3 is downregulated. That such regulation may exist is indicated by the inhibition of UMK3 at high substrate concentrations. In such a situation, the activity of UMK1 could be important for the recycling of NMPs from DNA/RNA breakdown or nucleoside salvaging. Although the Arabidopsis AMK-like UMKs are all ubiquitously expressed and have similar enzymatic activities, this work shows that they are not redundant or at best partially redundant, but fulfill specific biological functions.

U2 - 10.15488/16818

DO - 10.15488/16818

M3 - Doctoral thesis

CY - Hannover

ER -