The yeast YLR209c (PNP1) gene encodes a protein highly similar to purine nucleoside phosphorylases. This protein specifically metabolized inosine and guanosine. Disruption of PNP1 led to inosine and guanosine excretion in the medium, thus showing that PNP1 plays an important role in the metabolism of these purine nucleosides in vivo.Purine salvage is a complex pathway allowing interconversion of bases, nucleosides, and nucleotides. In yeast, major attention has been paid to the conversion of bases into nucleotides by phosphoribosyltransferases (PRTs): adenine-PRT, hypoxanthine-guanine-PRT, and xanthine-PRT activities have been reported (19,20), and the cognate genes have been identified (1,5,6). Yeast purine nucleoside metabolism has received far less attention, and only very recently was the first yeast gene encoding a purine nucleoside metabolizing enzyme identified (11). This gene, named ADO1, encodes adenosine kinase allowing synthesis of AMP from adenosine. Although several other enzymatic activities involved in yeast purine nucleoside metabolism have been described in the past, the corresponding genes have not yet been identified. Enzymatic activities responsible for the synthesis of inosine either from adenosine by adenosine deaminase (14) or from IMP by an IMP-specific 5Ј nucleotidase have been reported (8). Also, two distinct enzymatic activities (purine nucleoside hydrolase and purine nucleoside phosphorylase [PNP]) responsible for the degradation of inosine into hypoxanthine have been reported (7). The latter two enzymes catalyze the conversion of nucleosides to bases, although through distinct enzymatic mechanisms: (i) for nucleoside hydrolase, nucleoside ϩ H 2 O3base ϩ ribose and (ii) for nucleoside phosphorylase, nucleoside ϩ P i 3base ϩ ribose-1P.As a further step toward understanding yeast purine nucleoside metabolism, we searched for open reading frames (ORFs) in the complete yeast genome sequence that would encode candidate PNP. We found an uncharacterized ORF (YLR209c) that encodes a putative polypeptide highly similar to human and bovine PNP (Fig. 1). This enzyme has been thoroughly studied, and the three-dimensional structures of the trimeric human and bovine PNPs have been solved (3, 10). Important residues for substrate binding and catalysis have been identified (4, 12), all of which (except Val263) are conserved in the yeast enzyme (shown by asterisks in Fig. 1).To gain insight into the precise function of the yeast ORF YLR209c, the protein encoded by this ORF was tagged with 10 histidine residues at its N terminus and expressed in Escherichia coli. The PNP expression plasmid was constructed as follows. The YLR209c ORF was amplified by PCR from S288c genomic DNA with the following synthetic oligonucleotides: 359 (5Ј-CGATGCTCGAGATGAGTGATATCTTGAACGT-3Ј) and 360 (5Ј-GGACCCGGGTTATAATTCCCCCATTAC GG-3Ј). The amplification product was then digested with XhoI and SmaI and inserted in the pJC20-HisN vector (15) digested with XhoI and SmaI. The BL21(DE3) E. coli strain carrying the resulting plasmi...
In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.
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