Urh1p is a uridine-cytidine N-ribohydrolase that catalyzes the cleavage of N-glycosidic bonds in uridine, cytidine, and deoxycytidine, yielding ribose and the respective base (1, 3). Urh1p is also able to cleave bonds in the prodrugs 5-fluorouridine and 5-fluorocytidine, but is not able to use inosine, adenosine, guanosine, or thymidine as substrates (3, 1). Urh1p activity is crucial for recycling pyrimidine deoxy- and ribonucleosides via the pyrimidine nucleotide salvage and deoxynucleotide salvage pathways (3).

URH1 displays similarity to genes in other organisms, including two hypothetical genes from Schizosaccharomyces pombe (SPBC1683.06c and SPAC17G8.02), an inosine/uridine-preferring nucleosidase from the protozoan parasite Crithidia fasciculata (IUNH), three ribohydrolases from E. coli (RihA, RihB, RihC), the nonspecific nucleoside hydrolase of Leishmania major, and the nonspecific nucleoside hydrolase from the parasitic organism Trypanosoma brucei (3, 1).

urh1 null mutants are viable, and can utilize uridine or cytosine, but not cytidine, as the sole source of pyrimidines (3, 1). urh1 urk1 double mutants and ura3 urk1 urh1 triple mutants are unable to utilize cytidine or uridine as the sole source of pyrimidines (3, 1). Expression of the IUNH inosine/uridine-preferring nucleosidase from C. fasciculata in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of human uridine phosphorylase (UP), suggesting that yeast strains expressing protozoan N-ribohydrolases, or host phosphorylases, may be useful tools in drug screens for specific inhibitors (1).

About NAD biosynthesis -- de novo and salvage pathways

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for cellular redox reactions and energy metabolism. NAD also has been shown to be an important substrate in a variety of biological processes, including transcriptional regulation, DNA repair, calcium-dependent signaling pathways, calorie-restriction-mediated life-span extension and age-associated diseases (5, 6). NAD appears to affect these processes by regulating the Sir2p family of NAD-dependent deacetylases (Sirtuins) (6).

There are a number of pathways for NAD biosynthesis. In yeast and most other organisms, the two major pathways are de novo synthesis of NAD (the de novo pathway) and regeneration of NAD from its nicotinamide degradation products (the NAD salvage pathway) (7, 6). NAD is synthesized de novo from tryptophan via kynurenine (7). In this pathway tryptophan is converted to nicotinic acid mononucleotide (NaMN) in 6 enzymatic steps (catalyzed by Bna1-2p, and Bna4-7p) and one non-enzymatic step (7). At NaMN the de novo pathway converges with the NAD salvage pathway and the last two steps to NAD are shared (7, 6). In the yeast NAD salvage pathway, the vitamin precursors nicotinamide and nicotinic acid are converted to NaMN, the point of convergence with the de novo pathway (6). The steps from nicotinic acid to NAD were elucidated by Preiss and Handler and are sometimes referred to as the Preiss-Handler pathway (as reviewed in 8). Yeast can also import extracellular nicotinic acid extracellular nicotinic acid into the cell by the permease Tna1p and then convert it to NAD via the Preiss-Handler pathway (6).

There are four additional pathways for synthesizing NAD in yeast: two salvage pathways from the vitamin precursor nicotinamide riboside (NR) and two salvage pathways from nicotinic acid riboside (NaR) (8, 4, 9). Only one of these pathways, the NR salvage pathway I, is independent of the NAD salvage pathway. In the NR salvage pathway I, NR is phosphorylated to nicotinamide mononucleotide by the kinase Nrk1p, and then adenylated to NAD by Nma1p or Nma2p (8). In the NR salvage pathway II, the hydrolase Urh1p or the phosphorylase Pnp1p split NR into a ribosyl product and nicotinamide, which subsequently is converted to NAD via the NAD salvage pathway (4). The initial steps in the NaR salvage pathways I and II are similar to those of the NR salvage pathways I and II and are catalyzed by the same enzymes, respectively. In the NaR salvage pathway I, Nrk1p phosphorylates NaR to NaMN, which subsequently is converted to NAD via the enzymes shared by the de novo and NAD salvage pathways (9). In the NaR salvage pathway II, Urh1p or Pnp1p split NR into a ribosyl product and nicotinic acid, which is first converted to NaMN and then is converted similarly to NAD (9).

Last updated: 2005-11-30