Structure of the gene encoding phosphoribosylpyrophosphate synthetase (prsA) in Salmonella typhimurium

Abstract

The Salmonella typhimurium gene prsA, which encodes phosphoribosylpyrophosphate synthetase, has been cloned, and the nucleotide sequence has been determined. The amino acid sequence derived from the S. typhimurium gene is 99% identical to the derived Escherichia coli sequence and 47% identical to two rat isozyme sequences. Strains containing plasmid-borne prsA have been used to overproduce and purify the enzyme. The promoter for the S. typhimurium prsA gene was identified by deletion analysis and by similarity to the promoter for the E. coil prsA gene. The location of the prsA promoter results in a 416-base-pair 5' untranslated leader in the prsA transcript, which was shown by deletion to be necessary for maximal synthesis of phosphoribosylpyrophosphate synthetase. The S. typhimurium leader contains a 115-base-pair insert relative to the E. coli leader. The insert appears to have no functional significance.The enzyme phosphoribosylpyrophosphate (PRPP) synthetase catalyzes a reaction at a key junction in intermediary metabolism. The enzyme diverts ribose 5-phosphate from energy generation by the pentose phosphate pathway to biosynthesis via the intermediate PRPP. PRPP is utilized in the biosynthesis of pyridine nucleotide coenzymes, the amino acids histidine and tryptophan, and the pyrimidine and purine nucleotides. From 70 to 80% of the carbon flow through PRPP synthetase is directed to nucleotide and nucleic acid synthesis (15).The amount of PRPP synthetase activity in enteric bacteria is mediated at two levels: enzyme inhibition and regulation of synthesis. ADP is a potent inhibitor which binds at an allosteric site as well as competitively with ATP at the active site (5, 22). The ADP inhibition of PRPP synthetase at the allosteric site requires occupation of the active site. Thus, in the presence of ribose 5-phosphate, the activity of PRPP synthetase is mediated by the ratio of ADP to ATP. A pyrimidine, probably UDP or UTP, represses the synthesis of the enzyme 2-to 10-fold (17,26). A Salmonella typhimurium rpoBC mutant which showed derepressed levels of aspartate transcarbamylase (pyrBI) and orotate phosphoribosyltransferase (pyrE), enzymes that have been shown to be regulated by attenuation mechanisms, also had a derepressed level of PRPP synthetase (8). This observation suggests that an attenuation mechanism may also function to regulate the gene encoding PRPP synthetase (prsA).The catalytic mechanism of S. typhimurium PRPP synthetase has been studied extensively, whereas the genes encoding the Escherichia coli and rat enzymes have been cloned and sequenced (6,7,23). To initiate molecular genetic studies and to examine the regulation of S. typhimurium PRPP synthetase expression, the gene encoding the S. typhimurium PRPP synthetase has been cloned and the nucleotide sequence has been determined. MATERIALS AND METHODSMicrobiology and molecular biology. The strains used are listed in Table 1. Strain SB139 was produced from TR5878 * Corresponding author. and SB179 was produced from JL1002 by transduction with ...

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase

“…When the sequences of the rat (31), human (21), Bacillus subtilis (19), S. typhimurium (4), and E. coli (12) PRPP synthetases are aligned, it is evident that the site of the mutation in prs-100 (Arg-78) is not conserved. This residue is an Arg in the bacterial PRPP synthetases, but is replaced by Ile in the mammalian enzymes.…”

“…Purification of PRPP synthetase. Wild-type PRPP synthetase from strain SB139 containing plasmid pBS201 was purified as described by Bower et al (4).…”

Characterization of the hemA-prs region of the Escherichia coli and Salmonella typhimurium chromosomes: identification of two open reading frames and implications for prs expression

“…The first of these (102 bp) is found in the leader before ORFl and the second (115 bp) immediately follows the termination codon of ORF2, but precedes the prs gene. This latter insert was previously described by Bower et al (1988). Both ORFl and ORF2 are transcribed in a direction opposite of hemA, apparently from the promoter previously suggested by Verkamp & Chelm (1989), labelled P, in Figs 1 and 2.…”

“…The gene encoding phosphoribosylpyrophosphate (PRPP) synthetase b r s ) has been cloned and sequenced from both Escherichia coli (Hove-Jensen, 1985 ;HoveJensen et al, 1986) and Salmonella typhimurium (Bower et al, 1988); prs transcripts have a long 5' leader, 302 bp in E. coli (Hove-Jensen et al, 1986) and 417 bp in S. typhimurium (Bower et al, 1988). Roles for this leader have been suggested, but no clear function has been established.…”

Cloning and characterization of DjPRPS gene in freshwater planarian Dugesia japonica

“…It has been reported that there is a single-copy PRPS gene in E. coli [35], Bacillus subtilis [36] and Salmonella typhimurium [37], two in rat [38,24], at least three in man …”