Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine, Pyrimidine, and Cytokinin Ribosides  

Abstract

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.

Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

“…Hereto, we used the coordinates of inosine from the crystal structure of the YeiK-inosine complex (PDB code: 3B9X). In this structure inosine is bound in a conformation with a 0 4 0 -C 1 0 -N 9 -C 4 dihedral angle of 2528. 33 The calculation resulted in 20 inosine conformations docked in the CeNH active site.…”

“…are Ca 2+ ‐containing metalloenzymes that hydrolyze the N‐glycosidic bond of β‐ribonucleosides . They are widespread in nature, and representatives from the three domains of life, including eubacteria, archaea, yeast, protozoa, plants, insects, and mesozoans, have been characterized in different levels of detail . However, no representatives are present in humans.…”

“…2 They are widespread in nature, and representatives from the three domains of life, including eubacteria, archaea, yeast, protozoa, plants, insects, and mesozoans, have been characterized in different levels of detail. [4][5][6][7][8][9][10][11][12][13][14][15][16] However, no representatives are present in humans. Most attention has so far been devoted to the NHs from parasitic protozoa, including different Leishmania and Trypanosoma species.…”

Cytokinin signaling: from the ER or from the PM? That is the question!

“…The model predicts a buffering role of cytokinin nucleotides (tZRP) in the cytoplasm from which LOG enzymes catalyze the formation of biologically active tZ (Kuroha et al ., ). tZ may also be formed from tZR by nucleoside ribohydrolases (NRH) (Chen & Kristopeit, ; Kopečná et al ., ). tZ is the biologically active metabolite that penetrates the ER lumen and activates the ER‐located receptors.…”

“…LOG enzymes play the central role in generating from tZRP – which appears to be a metabolic buffer – biologically active tZ in the cytoplasm (Kurakawa et al ., ; Kuroha et al ., ; Tokunaga et al ., ). The functional relevance of an alternative, the direct formation of active bases from ribosides by nucleoside ribohydrolases, is less clear (Chen & Kristopeit, ; Kopečná et al ., ). The tZ released by LOG enzymes may penetrate into the ER lumen (Le Gall et al ., ; Lizák et al ., ) to trigger signaling from ER‐located cytokinin receptors.…”