Phosphoribosylpyrophosphate synthetase (PrsA) variants alter cellular pools of ribose 5-phosphate and influence thiamine synthesis in Salmonella enterica
Phosphoribosylamine (PRA) is the first intermediate in the common purine/thiamine biosynthetic pathway and is primarily synthesized by the product of the purF gene, glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (E.C. 2.4.2.14). Past genetic and biochemical studies have shown that multiple mechanisms for the synthesis of PRA independent of PurF are present in Salmonella enterica. Here, we describe mutant alleles of the essential prsA gene, which encodes PRPP synthetase (E.C.2.7.6.1), that allow PurF-independent thiamine synthesis. The mutant alleles resulted in reduced PrsA activity in extracts, caused nutritional requirements indicative of PRPP limitation and allowed non-enzymic formation of PRA due to a build-up of ribose 5-phosphate (R5P). These results emphasize the balance that must be reached between pathways competing for the same substrate to maintain robustness of the metabolic network.
BalchW. E.,
WolfeR. S.1976; New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid-dependent growth of Methanobacterium ruminantium in a pressurized atmosphere. Appl Environ Microbiol 32:781–791
BeckB. J.,
ConnollyL. E.,
De Las PenasA.,
DownsD. M.1997; Evidence that rseC, a gene in the rpoE cluster, has a role in thiamine synthesis in Salmonella typhimurium. J Bacteriol 179:6504–6508
CreightonT. E.1968; The nonenzymatic preparation in solution of N-(5-phosphoribosyl) anthranilic acid, an intermediate in tryptophan biosynthesis. J Biol Chem 243:5605–5609
DatsenkoK. A.,
WannerB. L.2000; One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
Enos-BerlageJ. L.,
DownsD. M.1996; Involvement of the oxidative pentose phosphate pathway in thiamine biosynthesis in Salmonella typhimurium. J Bacteriol 178:1476–1479
Enos-BerlageJ. L.,
LangendorfM. J.,
DownsD. M.1998; Complex metabolic phenotypes caused by a mutation in yjgF, encoding a member of the highly conserved YER057c/YjgF family of proteins. J Bacteriol 180:6519–6528
EriksenT. A.,
KadziolaA.,
BentsenA. K.,
HarlowK. W.,
LarsenS.2000; Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase. Nat Struct Biol 7:303–308
GralnickJ.,
DownsD.2001; Protection from superoxide damage associated with an increased level of the YggX protein in Salmonella enterica. Proc Natl Acad Sci U S A 98:8030–8035
GutnickD.,
ClvoJ. M.,
KlopotowaskiT.,
AmesB. N.1969; Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT2. J Bacteriol 100:215–219
HartmanP. E.,
HartmanZ.,
StahlR. C.1971; Classification and mapping of spontaneous and induced mutations in the histidine operon of Salmonella. Adv Genet 16:1–34
Hove-JensenB.1985; Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. Mol Gen Genet 201:269–276
Hove-JensenB.1988; Mutation in the phosphoribosylpyrophosphate synthetase gene ( prs) that results in simultaneous requirements for purine and pyrimidine nucleosides, nicotinamide nucleotide, histidine, and tryptophan in Escherichia coli. J Bacteriol 170:1148–1152
JensenK. F.1983; Metabolism of5-phosphoribosyl 1-pyrophosphate (PRPP) in Escherichia coli and Salmonella typhimurium. In Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms pp 1–26 Edited by
Munch-PetersenA.
London: Academic Press;
PetersenL.,
Enos-BerlageJ.,
DownsD. M.1996; Genetic analysis of metabolic cross-talk and its impact on thiamine synthesis in Salmonella typhimurium. Genetics 143:37–44
PostD. A.,
SwitzerR. L.1991; prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase. J Bacteriol 173:1978–1986
RamosI.,
VivasE. I.,
DownsD. M.2008; Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo. J Bacteriol 190:815–822
RobertsG. P.1978Isolation and Characterization of Informational Suppressors in Salmonella Typhimurium Berkeley, CA: University of California; Berkeley:
SchmiegerH.1972; Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet 119:75–88
ShimizuK.2004; Metabolic flux analysis based on 13C-labeling experiments and integration of the information with gene and protein expression patterns. Adv Biochem Eng Biotechnol 91:1–49
WayJ. C.,
DavisM. A.,
MorisatoD.,
RobertsD. E.,
KlecknerN.1984; New Tn 10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene 32:369–379
WillemoesM.,
NilssonD.,
Hove-JensenB.1996; Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichiacoli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding. Biochemistry 35:8181–8186
YenR. C.,
AdamsW. B.,
LazarC.,
BeckerM. A.1978; Evidence for X-linkage of human phosphoribosylpyrophosphate synthetase. Proc Natl Acad Sci U S A 75:482–485
Phosphoribosylpyrophosphate synthetase (PrsA) variants alter cellular pools of ribose 5-phosphate and influence thiamine synthesis in Salmonella enterica