STRUCTURE-FUNCTION STUDY ON MAIZE NUCLEOSIDE N-RIBOHYDROLASE FAMILY

Martina Tylichová¹, David Kopečný¹, Armelle Vigouroux², Radka Končitíková, Hanna Blaschke³, Klaus von Schwartzenberg³, Solange Moréra²

¹Department of Protein Biochemistry and Proteomics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic; ²Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, F-91198 Gif-sur-Yvette Cedex, France; ³Biozentrum Klein Flottbek, Universität Hamburg, D–22609 Hamburg, Germany

Nucleoside hydrolases also called nucleoside N-ribohydrolases (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. The recycling of nucleosides and nucleobases is also known as the salvage pathway and together with de novo synthesis and catabolism of purines and pyrimidines appears in the overall nucleotide metabolism. NRHs belong to metalloproteins. All enzymes characterized so far impose a strict specificity for the ribose moiety, but they exhibit variability in their preferences for the nucleobase. NRHs were initially identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia and Leishmania. These organisms rely on the import and salvage of nucleotide derivatives as they do not possess enzymes for nucleotide de novo synthesis. In plants, only NRH gene family in Arabidopsis thaliana was deeply analyzed so far. In maize (Zea mays), five related genes can be found. Two paralog genes of ZmNRH1a and ZmNRH1b are localized to chromosome 8 and 3. Other two paralogs, genes of ZmNRH2a and ZmNRH2b, lie at chromosome 4 and 1.The ZmNRH3 gene is localized to chromosome 2. All five genes from Zea mays were cloned and expressed in E. coli. The substrate specificity of the recombinant enzymes was analyzed and significant differences were observed. While both ZmNRH2a and ZmNRH2b preferentially catalyze the conversion of pyrimidine nucleosides, ZmNRH3 hydrolyzes purine nucleosides including inosine and xanthosine. The ZmRNH3 was crystallized and then diffraction data were collected at 2.5 Ǻ resolution on Proxima 1 beamline at SOLEIL synchrotron (Saclay, France). The crystals belong to the orthorhombic space group P2₁2₁2₁ and contain one dimer per asymmetric unit. The enzyme comprises four Asp residues in a conserved sequence motif DXDXXXDD at the N terminus. The first aspartate residue is involved in catalysis while the second and fourth aspartate are involved in the coordination of a calcium ion at the active site. Finally, ZmNRHs also cleave cytokinin ribosides to the corresponding bases indicating that the enzyme could contribute to cytokinin homeostasis. Cytokinins (N⁶-substituted adenine or adenosine derivatives) are the plant hormones regulating cell division as well as a large number of developmental events, such as shoot and root branching, leaf development, and chloroplast ripening.

This work was supported by grant P501/11/1591 from the Czech Science Foundation.