Uridine-ribohydrolase is a key regulator in the uridine degradation pathway of Arabidopsis.
Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding K(m) values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter beta-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development.
Jung
Benjamin
B
Abteilung Pflanzenphysiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, D-67663 Kaiserslautern, Germany.
Flörchinger
Martin
M
Kunz
Hans-Henning
HH
Traub
Michaela
M
Wartenberg
Ruth
R
Jeblick
Wolfgang
W
Neuhaus
H Ekkehard
HE
Möhlmann
Torsten
T
D000595:Amino Acid Sequence; D000872:Anthocyanins / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014529:Uridine / Q000378:metabolism*
D000872:Anthocyanins; D029681:Arabidopsis Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; C023277:uridine nucleosidase; D001120:Arginine; D009699:N-Glycosyl Hydrolases; C579244:URH1 protein, Arabidopsis; D009584:Nitrogen; D014529:Uridine