Study on active site of nucleoside N-ribohydrolases from Zea mays

Eva Hájková1, Armelle Vigouroux2, Radka Končitíková1, Martina Kopečná1, David Zalabák3, Ondřej Novák4, Ondřej Plíhal3, Solange Moréra2, David Kopečný1

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

2Institute for Integrative Biology of the Cell, CNRS-CEA-Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

3Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, Czech Republic

4Laboratory of Growth Regulators, Institute of Experimental Botany ASCR & Palacký University, Olomouc, Czech Republic

 

Purine and pyrimidine nucleosides are hydrolyzed by nucleoside N-ribohydrolases (NRHs, E.C. 3.2.2.-). These calcium-dependent enzymes catalyze a cleavage of the N-glycosidic bond in nucleosides to enable the recycling of the nucleobases and ribose. NRHs impose a strict specificity for the ribose moiety while residues interacting with the nucleobase highly vary. There are at least two subclasses of NRHs in plants, which belong to a class of nonspecific inosine/uridine NRHs. They differ in their substrate specificity and preferences towards inosine and xanthosine (subclass I) or uridine and xanthosine (subclass II). We performed a crystallographic study combined with site-directed mutagenesis and kinetic analyses to study nucleoside binding sites in two maize NRHs representing both subclasses, namely ZmNRH2b and ZmNRH3. Crystal structures of ZmNRH2b apoform and its complexes were solved up to 1.75 Å resolution and they were further compared with the structure of ZmNRH3. A role of several active-site residues was deeply studied in both enzymes detail and certain mutations were found to increase the hydrolysis of cytokinin ribosides. We further analyzed spatial and temporal expression of all five ZmNRH genes present in maize as well as a transient expression of ZmNRH-GFP fusion proteins in maize protoplasts. We also constructed and selected homozygous dexamethasone-inducible ZmNRH overexpressor lines (for all five maize NRH genes) in Arabidopsis thaliana to analyze the enzyme function in planta and measured levels of purine, pyrimidine and cytokinin metabolites. Finally, our experiments proved that NRHs metabolize also cytotoxic metabolites like 5-fluorouridine and 2-chloroadenosine.

 

This work was supported by the grant 15-22322S from the Czech Science Foundation, the MOBILITY grant 7AMB17DE009 and the grant LO1204 from the National Program of Sustainability I by the Ministry of Education, Youth and Sports, Czech Republic and internal grants IGA_PrF_2017_016 and IGA_PrF_2018_033 from Palacký University Olomouc.