Publications

• Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae.

  Hernando Y., Carter A.T., Parr A., Hove-Jensen B., Schweizer M.

  The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible ... >> More

  The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es). << Less

  J. Biol. Chem. 274:12480-12487(1999) [PubMed] [EuropePMC]

• Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii.

  Kadziola A., Jepsen C.H., Johansson E., McGuire J., Larsen S., Hove-Jensen B.

  The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and ... >> More

  The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K(m) values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V(max) value as 2.20 mmol (minxmg of protein)(-1). ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M.jannaschii enzyme. The M.jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B.subtilis PRPP synthase. Soaking of the crystals with 5'-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases. << Less

  J. Mol. Biol. 354:815-828(2005) [PubMed] [EuropePMC]

• Regulation and mechanism of phosphoribosylpyrophosphate synthetase. I. Purification and properties of the enzyme from Salmonella typhimurium.

  Switzer R.L.

  J. Biol. Chem. 244:2854-2863(1969) [PubMed] [EuropePMC]

• Mycobacterium tuberculosis phosphoribosylpyrophosphate synthetase: biochemical features of a crucial enzyme for mycobacterial cell wall biosynthesis.

  Lucarelli A.P., Buroni S., Pasca M.R., Rizzi M., Cavagnino A., Valentini G., Riccardi G., Chiarelli L.R.

  The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, ... >> More

  The selection and soaring spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Currently, there is an urgent need for new drugs for tuberculosis treatment, with novel mechanisms of action and, moreover, the necessity to identify new drug targets. Mycobacterial phosphoribosylpyrophosphate synthetase (MtbPRPPase) is a crucial enzyme involved in the biosynthesis of decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis. Moreover, phosphoribosylpyrophosphate, which is the product of the PRPPase catalyzed reaction, is the precursor for the biosynthesis of nucleotides and of some amino acids such as histidine and tryptophan. In this context, the elucidation of the molecular and functional features of MtbPRPPase is mandatory. MtbPRPPase was obtained as a recombinant form, purified to homogeneity and characterized. According to its hexameric form, substrate specificity and requirement of phosphate for activity, the enzyme proved to belong to the class I of PRPPases. Although the sulfate mimicked the phosphate, it was less effective and required higher concentrations for the enzyme activation. MtbPRPPase showed hyperbolic response to ribose 5-phosphate, but sigmoidal behaviour towards Mg-ATP. The enzyme resulted to be allosterically activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+) and Cu(2+) but, differently from other characterized PRPPases, it showed a better affinity for the Mn(2+) and Cu(2+) ions, indicating a different cation binding site geometry. Moreover, the enzyme from M. tuberculosis was allosterically inhibited by ADP, but less sensitive to inhibition by GDP. The characterization of M. tuberculosis PRPPase provides the starting point for the development of inhibitors for antitubercular drug design. << Less

  PLoS ONE 5:E15494-E15494(2010) [PubMed] [EuropePMC]

• Biochemical characterization of the Mycobacterium tuberculosis phosphoribosyl-1-pyrophosphate synthetase.

  Alderwick L.J., Lloyd G.S., Lloyd A.J., Lovering A.L., Eggeling L., Besra G.S.

  Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane b ... >> More

  Mycobacterium tuberculosis arabinogalactan (AG) is an essential cell wall component. It provides a molecular framework serving to connect peptidoglycan to the outer mycolic acid layer. The biosynthesis of the arabinan domains of AG and lipoarabinomannan (LAM) occurs via a combination of membrane bound arabinofuranosyltransferases, all of which utilize decaprenol-1-monophosphorabinose as a substrate. The source of arabinose ultimately destined for deposition into cell wall AG or LAM originates exclusively from phosphoribosyl-1-pyrophosphate (pRpp), a central metabolite which is also required for other essential metabolic processes, such as de novo purine and pyrimidine biosyntheses. In M. tuberculosis, a single pRpp synthetase enzyme (Mt-PrsA) is solely responsible for the generation of pRpp, by catalyzing the transfer of pyrophosphate from ATP to the C1 hydroxyl position of ribose-5-phosphate. Here, we report a detailed biochemical and biophysical study of Mt-PrsA, which exhibits the most rapid enzyme kinetics reported for a pRpp synthetase. << Less

  Glycobiology 21:410-425(2011) [PubMed] [EuropePMC]

• Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase.

  Willemoes M., Hove-Jensen B.

  The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP a ... >> More

  The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP and (+)-1-alpha,2-alpha,3-alpha-trihydroxy-4-beta-cyclopentanemethanol 5-phosphate, respectively, the binding of Mg2+ and the substrates were determined to occur via a steady state ordered mechanism in which Mg2+ binds to the enzyme first and ribose 5-phosphate binds last. Mg2+ binding to the enzyme prior to the binding of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands Mg x ATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate. Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by Mg x ADP indicated that Mg x ADP binding to the allosteric site may occur in competition with enzyme bound Mg2+. Ligand binding studies showed that 1 mol of Mg x ATP was bound per mol of phosphoribosyl diphosphate synthetase subunit, which indicated that the allosteric sites of the multimeric enzyme were not made up by inactive catalytic sites. << Less

  Biochemistry 36:5078-5083(1997) [PubMed] [EuropePMC]

• Phosphoribosyl diphosphate (PRPP): biosynthesis, enzymology, utilization, and metabolic significance.

  Hove-Jensen B., Andersen K.R., Kilstrup M., Martinussen J., Switzer R.L., Willemoes M.

  Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bond ... >> More

  Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species. << Less

  Microbiol. Mol. Biol. Rev. 81:E00040-E00040(2017) [PubMed] [EuropePMC]

• PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae.

  Carter A.T., Beiche F., Hove-Jensen B., Narbad A., Barker P.J., Schweizer L.M., Schweizer M.

  In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PR ... >> More

  In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. << Less

  Mol. Gen. Genet. 254:148-156(1997) [PubMed] [EuropePMC]

• Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop.

  Hove-Jensen B., Bentsen A.K., Harlow K.W.

  Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an ... >> More

  Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype enzyme. Kinetic characterization showed that the V(max) values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced, respectively, compared to the wildtype enzyme. The K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state. << Less

  FEBS J. 272:3631-3639(2005) [PubMed] [EuropePMC]

• Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae: combinatorial expression of the five PRS genes in Escherichia coli.

  Hove-Jensen B.

  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 (Deltaprs) showed that a single PRS g ... >> More

  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 (Deltaprs) 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 Pi, to be temperature-sensitive, and inhibited by ADP. << Less

  J. Biol. Chem. 279:40345-40350(2004) [PubMed] [EuropePMC]

• Regulation and mechanism of phosphoribosylpyrophosphate synthetase. 3. Kinetic studies of the reaction mechanism.

  Switzer R.L.

  J. Biol. Chem. 246:2447-2458(1971) [PubMed] [EuropePMC]

• Inactivation of Escherichia coli phosphoribosylpyrophosphate synthetase by the 2',3'-dialdehyde derivative of ATP. Identification of active site lysines.

  Hilden I., Hove-Jensen B., Harlow K.W.

  The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2',3'-dialdehyde ATP (oATP). The reaction displayed complex saturation kinetics with respect to oATP with an apparent KD of approximately 0.8 mM ... >> More

  The enzyme 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase from Escherichia coli was irreversibly inactivated on exposure to the affinity analog 2',3'-dialdehyde ATP (oATP). The reaction displayed complex saturation kinetics with respect to oATP with an apparent KD of approximately 0.8 mM. Reaction with radioactive oATP demonstrated that complete inactivation of the enzyme corresponded to reaction at two or more sites with limiting stoichiometries of approximately 0.7 and 1.3 mol of oATP incorporated/mol of PRPP synthetase subunit. oATP served as a substrate in the presence of ribose-5-phosphate, and the enzyme could be protected against inactivation by ADP or ATP. Isolation of radioactive peptides from the enzyme modified with radioactive oATP, followed by automated Edman sequencing allowed identification of Lys181, Lys193, and Lys230 as probable sites of reaction with the analog. Cysteine 229 may also be labeled by oATP. Of these four residues, Lys193 is completely conserved within the family of PRPP synthetases, and Lys181 is found at a position in the sequence where the cognate amino acid (Asp181) in human isozyme I PRPP synthetase has been previously implicated in the regulation of enzymatic activity. These results imply a functional role for at least two of the identified amino acid residues. << Less

  J. Biol. Chem. 270:20730-20736(1995) [PubMed] [EuropePMC]

• Binding of the substrates and the allosteric inhibitor adenosine 5'-diphosphate to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium.

  Gibson K.J., Schubert K.R., Switzer R.L.

  The binding of the substrates, ATP and ribose-5-P, and the most effective inhibitor, ADP, to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium was characterized using equilibrium dialysis of these compounds labeled with 32P. In the absence of ribose-5-P, ATP, ADP, and the ATP anal ... >> More

  The binding of the substrates, ATP and ribose-5-P, and the most effective inhibitor, ADP, to phosphoribosylpyrophosphate synthetase from Salmonella typhimurium was characterized using equilibrium dialysis of these compounds labeled with 32P. In the absence of ribose-5-P, ATP, ADP, and the ATP analogue alpha,beta-methylene ATP each bind cooperatively with half-saturation at 50 to 90 microM and Hill coefficients of 1.5 to 2. We propose that all three compounds bind at the same set of sites, which are presumably the active sites. When ribose-5-P was added, methylene ATP and ADP binding at these sites became tighter (Kd approximately 3 to 6 microM at 10 mM ribose-5-P) and lost its cooperativity. In the presence of ribose-5-P, ADP, but not methylene ATP, bound to a second site with half-saturation at approximately 150 microM and a Hill coefficient greater than 3. This result confirms the existence of an allosteric ADP site, which was previously postulated from kinetic studies (Switzer, R. L., and Sogin, D. C. (1973) J. Biol. Chem. 248, 1063-1073). Binding of ribose-5-P could not be detected in the absence of nucleotides, but it was readily measured in their presence. The apparent Kd of ribose-5-P varied from greater than 1 mM to approximately 5 microM as the concentration of either ADP or methylene ATP was increased from 0 to 2 mM. Inhibition of the enzyme by action of ADP at both active and allosteric sites could be observed kinetically. << Less

  J. Biol. Chem. 257:2391-2396(1982) [PubMed] [EuropePMC]

• Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis.

  Arnvig K., Hove-Jensen B., Switzer R.L.

  Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli delta prs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino aci ... >> More

  Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli delta prs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid sequence (14 residues) were in complete agreement with expectations from the nucleotide sequence of the prs gene. The Mr of the native enzyme (280,000 +/-10,000) was consistent with an octameric quaternary structure. No tendency toward multiple states of aggregation of the enzyme was seen. The purified enzyme required Mg2+ and inorganic phosphate for activity; Mn2+ supported only 30% the activity seen with Mg2+. Michaelis constants for ATP and ribose 5-phosphate (Rib5P) were 0.66 mM and 0.48 mM, respectively. Of several end products tested, only ADP was strongly inhibitory; GDP was a weak inhibitor. ADP inhibition displayed homotropic cooperativity and was enhanced by increasing saturation of the enzyme with ATP. These observations strongly suggest a specific allosteric site for ADP binding. A comparison of physical and kinetic properties of bacterial and mammalian PPRibP synthetases is presented. << Less

  Eur. J. Biochem. 192:195-200(1990) [PubMed] [EuropePMC]

• Wild-type phosphoribosylpyrophosphate synthase (PRS) from Mycobacterium tuberculosis: a bacterial class II PRS?

  Breda A., Martinelli L.K., Bizarro C.V., Rosado L.A., Borges C.B., Santos D.S., Basso L.A.

  The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis ... >> More

  The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of P(i). ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of P(i) would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme. << Less

  PLoS ONE 7:E39245-E39245(2012) [PubMed] [EuropePMC]