title: 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.
authors.affiliation: Department of Protein Biochemistry and Proteomics, Center of the Region Haná for Biotechnological and Agricultural Research , Faculty of Science, Palacký University, CZ-783 71 Olomouc, Czech Republic; Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, CZ-783 71 Olomouc, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Kopecná
authors.forename: Hanna
authors.initials: H
authors.lastname: Blaschke
authors.forename: David
authors.initials: D
authors.lastname: Kopecny
authors.forename: Armelle
authors.initials: A
authors.lastname: Vigouroux
authors.forename: Radka
authors.initials: R
authors.lastname: Koncitíková
authors.forename: Ondrej
authors.initials: O
authors.lastname: Novák
authors.forename: Ondrej
authors.initials: O
authors.lastname: Kotland
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.forename: Solange
authors.initials: S
authors.lastname: Moréra
authors.forename: Klaus
authors.initials: K
authors.lastname: von Schwartzenberg
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D019068:Bryopsida / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D018360:Crystallography, X-Ray; D003583:Cytokinins / Q000737:chemistry / Q000378:metabolism; D055786:Gene Knockout Techniques; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009584:Nitrogen / Q000494:pharmacology; D010641:Phenotype; D010802:Phylogeny; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism*; D012263:Ribonucleosides / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D003313:Zea mays / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D003583:Cytokinins; D050505:Mutant Proteins; D011743:Pyrimidines; D012263:Ribonucleosides; D009699:N-Glycosyl Hydrolases; C030986:pyrimidine; D009584:Nitrogen