Family of plant nucleoside N-ribohydrolases

David Kopečný¹, Martina Kopečná¹, Radka Končitíková¹, Armelle Vigouroux², Klaus von Schwartzenberg³, Solange Moréra²

¹Department of Protein Biochemistry and Proteomics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Olomouc, Czech Republic

²Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France

³Biozentrum Klein Flottbek, Universität Hamburg, Germany

Nucleoside hydrolases or nucleoside N-ribohydrolases (NHs, NRHs, E.C. 3.2.2.-) are glycosidases that catalyze the excision of the N-glycosidic bond in nucleosides to allow recycling of the nitrogenous bases and ribose. NRHs are metalloproteins first identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia and Leishmania, which rely on the import and salvage of nucleotide derivatives. The enzyme comprises four Asp residues located in a conserved motif DXDXXXDD at the N terminus. These aspartates are involved in catalysis and coordination of a calcium ion at the active site. The binding of ribose moiety is highly conserved but the residues interacting with nucleobase highly vary. Therefore all NRHs characterized so far impose a strict specificity for the ribose moiety but they exhibit variability in their preferences for the nucleobase. We performed a comprehensive characterization of the NRH family in two model plants, Physcomitrella patens (PpNRH) and Zea mays (ZmNRH). The moss Physcomitrella patens represents the bryophytes, which can be regarded as being evolutionary basal terrestrial plant, and is suitable for use in developmental and metabolic studies, while Zea mays is an important model system for cereal crops. We identified 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. Both subclasses can hydrolyze plant hormones - cytokinin ribosides. Crystal structures of two purine NRHs, PpNRH1 and ZmNRH3, were solved. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies allowed us 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. To analyze the physiological role of the PpNRHs, single knockout mutants were generated. NRH deficiency caused delayed bud formation and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. NRH deficiency was accompanied by significant changes in the levels of purine, pyrimidine and cytokinin metabolites relative to those seen in the wild-type, illustrating the importance of these enzymes in nucleoside and cytokinin metabolism.

This work was supported by grant P501/11/1591 from the Czech Science Foundation and LO1204 from the Ministry of Education, Youth and Sports of the Czech Republic.