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 enzyme comprises four aspartate residues located at the N terminus, which are involved in catalysis and coordination of a calcium ion at the active site. The binding of ribose moiety is highly conserved and NRHs impose a strict specificity for the ribose moiety. In contrast, the residues interacting with nucleobase highly vary. 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. In this work, we combined a site-directed mutagenesis approach with kinetic and structural analyses to study nucleoside binding sites in two NRHs from maize (Zea mays), namely ZmNRH2b (GenBank accession number JQ594984) and ZmNRH3 (HQ825162). The first one is uridine specific while the latter one is purine specific nucleosidase. The crystal structures of both ZmNRHs were solved at 1.75 and 2.51 Å resolution, respectively, and confirm that enzymes from both subclasses are dimers. Five ZmNRH2b variants and seven ZmNRH3 variants were prepared and changes in their kinetics were studied in detail. Replacement of two active-site histidine residues by alanine leads 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, reduces the specific activity to 30% but increases hydrolysis of cytokinin ribosides compared to wild-type. Finally, replacement of active-site tyrosine and tryptophane residues alters substrate specificity for major substrates – xanthosine and uridine.
This work was supported by grant 15-22322S from the Czech Science Foundation and grant IGA_PrF_2014020 from Palacký University.