Heterooligomeric Phosphoribosyl Diphosphate Synthase of Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (⌬prs) showed that a single PRS gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no detectable PRPP synthase activity. In contrast PRPP could be detected in growing cells containing PRS1 PRS2, PRS1 PRS3, PRS5 PRS2, or PRS5 PRS4. These apparent conflicting results indicate that, apart from the PRS1 PRS3-specified enzyme, PRS-specified enzyme is functional in vivo but unstable when released from the cell. Certain combinations of three PRS genes appeared to produce an enzyme that is stable in vitro. Thus, extracts of strains harboring PRS1 PRS2 PRS5, PRS1 PRS4 PRS5, or PRS2 PRS4 PRS5 as well as extracts of strains harboring combinations with PRS1 PRS3 contained readily assayable PRPP synthase activity. The data indicate that although certain pairwise combinations of subunits produce an active enzyme, yeast PRPP synthase requires at least three different subunits to be stable in vitro. The activity of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5 was found to be dependent on P i , to be temperature-sensitive, and inhibited by ADP.is an important component of the metabolism of most organisms. PRPP is a precursor of the biosynthesis of purine, pyrimidine, and pyridine nucleotides, as well as of the amino acids tryptophan and histidine (1). The biosynthesis of PRPP is catalyzed by PRPP synthase (ATP: D-ribose 5-phosphate pyrophosphotransferase, EC 2.7.6.1), which is encoded by a PRS gene: ribose 5-phosphate ϩ ATP 3 PRPP ϩ AMP (2). Except for certain specialized mutants of Escherichia coli, all freeliving organisms contain at least one gene encoding PRPP synthase. These E. coli mutants require guanosine, uridine, histidine, tryptophan, and NAD (1, 3). The yeast Saccharomyces cerevisiae contains five PRPP synthase-homologous genes, PRS1-5, encoding PRPP synthase subunits 1-5, respectively. All five genes have been shown to be expressed (4 -6). Phylogenetic analysis revealed a close relationship of S. cerevisiae PRPP synthase subunits with PRPP synthases from other eukaryotic organisms, including human, rat, and Caenorhabditis elegans. S. cerevisiae PRPP synthase subunits 2, 3, and 4 resemble the "classical" class I PRPP synthases from E. coli, Bacillus subtilis, and human in length (311-355 amino aci...

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…This analysis furthermore showed that the simultaneous expression of PRS1 and PRS2 or the simultaneous expression of PRS1 and PRS4 resulted in PRPP synthase with very low activity in vivo, presumably too low to promote growth of S. cerevisiae Δprs3 Δprs4 Δprs5 or Δprs2 Δprs3 Δprs5 deletants. Although complementation of the E. coli Δprs allele by combinations of two PRS genes revealed the production of active PRPP synthase in vivo, in vitro PRPP synthase activity could be detected only in cell extract of the strain that expressed both PRS1 and PRS3 (82). The conclusions from this analysis are that active PRPP synthase requires either PRS1 or PRS5 in addition to at least one more PRS gene product, but not just any PRS gene product.…”

“…Little is known of the enzymatic properties of S. cerevisiae PRPP synthase activity. Some activity remains following dialysis of an S. cerevisiae crude extract against P i -free buffer and in assays under P i -free conditions, and only 18% of the activity remained when the enzyme was assayed in the presence of 2 mM ADP, relative to 100% in the absence of ADP (82). These properties differ from those of bacterial PRPP synthases.…”

“…Unlike most other cloned prs genes, the gene products of each of the five S. cerevisiae PRS genes have no PRPP synthase activity in vivo when their genes are harbored and expressed individually in an E. coli host strain (82). In contrast, the formation of active PRPP synthase requires the expression of two or more PRS genes, as established by deletion analysis of PRS genes in S. cerevisiae or by expression of PRS genes in E. coli.…”

Using Gene Essentiality and Synthetic Lethality Information to Correct Yeast and CHO Cell Genome-Scale Models

“…The inconsistency of this GPR was addressed in i AZ900 [ 8 ] and was modified to depict that any of the three PRS gene pairs ( PRS1 and PRS3 ), ( PRS2 and PRS5 ) or ( PRS4 and PRS5 ) is capable of encoding the sub-units required for catalyzing the reaction. However, it was later shown [ 68 ] that any of the five viable pairs (see Figure 4 ) need to be present for growth with one subunit containing an NHR (non-homologous region) and the other without one. Both PRS1 and PRS5 contain NHR while the rest do not.…”

Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

“…PRPP is also associated with cell integrity in Saccharomyces cerevisiae [15]. The yeast genome encodes five distinct prs genes, whose products are combined to form hetero-oligomeric catalytic active PRS, with possible role in plasma membrane stability [16]. In Corynebacteriaceae, such as mycobacteria, PRPP is a co-substrate for the synthesis of polyprenylphosphate-pentoses, which are the source of arabinosyl residues of arabinogalactan, component of the mycobacterial cell wall, and lipoarabinomannan, a highly immunogenic lipoglycan that is involved in modulating the host immune response [17], [18].…”