Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N-ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase.
Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.
Mitterbauer
Rudolf
R
Center of Applied Genetics, University of Agricultural Sciences, Muthgasse 18/5/66, A-1190 Vienna, Austria.
Karl
Thomas
T
Adam
Gerhard
G
D000595:Amino Acid Sequence; D000818:Animals; D003562:Cytidine / Q000378:metabolism*; D056890:Eukaryota / Q000201:enzymology; D005816:Genetic Complementation Test; D006801:Humans; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014529:Uridine / Q000378:metabolism*; D014543:Uridine Phosphorylase
D029701:Saccharomyces cerevisiae Proteins; D003562:Cytidine; D014543:Uridine Phosphorylase; D006867:Hydrolases; D009699:N-Glycosyl Hydrolases; C454867:URH1 protein, S cerevisiae; D014529:Uridine