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.
Inhibition of IspD leading to herbicidal activity has been ruled out as the mode of action for the hydroxytriazolopyrimidine class of herbicides. Additionally, tobacco plants overexpressing lycopene β-cyclase showed tolerance to amitrole, which indicates that this is the main herbicidal mode of action for amitrole. Results from the metabolic fate study of selected hydroxytriazolopyrimidines suggested that the herbicidal activity displayed by these compounds is due to amitrole production, which was confirmed when tobacco plants overexpressing lycopene β-cyclase also showed tolerance towards two triazolopyrimidines from this study. © 2016 Society of Chemical Industry.
We have characterized a new locus, BRA3, leading to deregulation of the yeast purine synthesis genes (ADE genes). We show that bra3 mutations are alleles of the GUK1 gene, which encodes GMP kinase. The bra3 mutants have a low GMP kinase activity, excrete purines in the medium, and show vegetative growth defects and resistance to purine base analogs. The bra3 locus also corresponds to the previously described pur5 locus. Several lines of evidence indicate that the decrease in GMP kinase activity in the bra3 mutants results in GMP accumulation and feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), encoded by the HPT1 gene. First, guk1 and hpt1 mutants share several phenotypes, such as adenine derepression, purine excretion, and 8-azaguanine resistance. Second, overexpression of HPT1 allows suppression of the deregulated phenotype of the guk1 mutants. Third, we show that purified yeast HGPRT is inhibited by GMP in vitro. Finally, incorporation of hypoxanthine into nucleotides is similarly diminished in hpt1 and guk1 mutants in vivo. We conclude that the decrease in GMP kinase activity in the guk1 mutants results in deregulation of the ADE gene expression by phenocopying a defect in HGPRT. The possible occurrence of a similar phenomenon in humans is discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.