Nucleosides and nucleobases can be recycled to nucleoside monophosphates, which is also known as salvage pathway preserving an energy, which would otherwise be needed for de novo synthesis of purine and pyrimidine derivatives. In plants, both uridine kinase (UK, E.C. 2.7.1.48) and uracil phosphoribosyl transferase act on the pyrimidine salvage pathway. In purine salvage, an important role has been shown for adenosine kinase (AK, E.C. 2.7.1.20) and adenine phosphoribosyltransferase. Purine and pyrimidine nucleosides are hydrolyzed by nucleoside N-ribohydrolases (NRHs, E.C. 3.2.2.-) to corresponding nitrogenous bases and ribose. There are two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine while the other is more active towards uridine and xanthosine.
In this work, we focused on purine metabolism and interconversion of plant hormones cytokinins, which are N6-substituted adenine/adenosine derivatives. We analyzed spatial and temporal expression of all five NRH and three ADK genes present in maize (Zea mays). Transcripts of ADK2 were the most abundant of all three ADKs in all organs and developmental stages. Transcripts of NRH1a, NRH2a and NRH3 genes are the most abundant in leaves while roots contain mainly NRH1b and NRH3 transcripts. Protein sequences of ADKs comprise ~ 340 amino acids and are monomeric. Those of NRHs encompass ~ 320 amino acids and enzymes are dimeric. NRHs impose a strict specificity for the ribose moiety. In contrast, the residues interacting with nucleobase highly vary. We combined a site-directed mutagenesis approach with kinetic and structural analyses to study nucleoside binding sites in two NRHs, namely NRH2b and NRH3. The crystal structures of both NRHs were solved at 1.75 and 2.51 Å resolution, respectively. Five NRH2b variants and seven NRH3 variants were studied in detail. Replacement of two active-site histidine residues by alanine led to two-fold lower specific activities. Replacement of a lysine residue, which protrudes into the active site from the second subunit and interacts with nucleobase, reduced the specific activity but increased hydrolysis of cytokinin ribosides isopentenyl adenosine (iPR) and trans-zeatin riboside (ZR). Replacement of the active-site tryptophan residue in NRH3 also increased hydrolysis of cytokinin ribosides.
This work was supported by grant 15-22322S from the Czech Science Foundation and grant IGA_PrF_2016_024 from Palacký University.