Recombinant Uracil Phosphoribosyltransferase from the ThermophileBacillus caldolyticus:Expression, Purification, and Partial Characterization

Jensen, Helle Kock; Mikkelsen, Nanna S.; Neuhard, Jan

doi:10.1006/prep.1997.0755

Cited by 21 publications

(18 citation statements)

References 24 publications

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“…The Michaelis constant for PRPP was also somewhat dependent on pH; the minimal value of 70 M was observed at pH 8.7 with larger values observed at both higher and lower pH. The K m values for PyrR-catalyzed UPRTase are in contrast to the values of about 50 M for PRPP and 2 M for uracil observed with the B. caldolyticus uppencoded UPRTase at pH 8.6 (19). We suggest that these kinetic differences between the pyrR-encoded UPRTase and the uppencoded UPRTase, which has much greater sequence similarity to other bacterial UPRTases (7), explain why the upp-encoded enzyme is the physiologically dominant UPRTase in B. subtilis (7): the latter enzyme has a much smaller Michaelis constant for uracil and is thus much more effective in uracil salvage.…”

Section: Fig 1 Esi-ms Analysis Of Purified Pyrrmentioning

confidence: 76%

Purification and Characterization of Bacillus subtilis PyrR, a Bifunctional pyr mRNA-binding Attenuation Protein/Uracil Phosphoribosyltransferase

Turner¹,

Bonner²,

Grabner

et al. 1998

Journal of Biological Chemistry

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Bacillus subtilis PyrR has been shown to mediate transcriptional attenuation at three separate sites within the pyrimidine nucleotide biosynthetic (pyr) operon. Molecular genetic evidence suggests that regulation is achieved by PyrR binding to pyr mRNA. PyrR is also a uracil phosphoribosyltransferase (UPRTase). Recombinant PyrR was expressed in Escherichia coli, purified to homogeneity, physically and chemically characterized, and examined with respect to both of these activities. Mass spectroscopic characterization of PyrR demonstrated a monomeric mass of 20,263 Da. Gel filtration chromatography showed the native mass of PyrR to be dependent on protein concentration and suggested a rapid equilibrium between dimeric and hexameric forms. The UPRTase activity of PyrR has a pH optimum of 8.2. The K m value for uracil is very pH-dependent; the K m for uracil at pH 7.7 is 990 ؎ 114 M, which is much higher than for most UPRTases and may account for the low physiological activity of PyrR as a UPRTase. Using an electrophoretic mobility shift assay, PyrR was shown to bind pyr RNA that includes sequences from its predicted binding site in the second attenuator region. Binding of PyrR to pyr RNA was specific and UMP-dependent with apparent K d values of 10 and 220 nM in the presence and absence of UMP, respectively. The concentration of UMP required for half-maximal stimulation of binding of PyrR to RNA was 6 M. The results support a model for the regulation of pyr transcription whereby termination is governed by the UMP-dependent binding of PyrR to pyr RNA and provide purified and characterized PyrR for detailed biochemical studies of RNA binding and transcriptional attenuation.The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of UMP and two additional cistrons encoding a uracil permease and the regulatory protein PyrR (1-4). On the basis of molecular genetic evidence it was proposed that PyrR regulates pyr expression through a transcriptional attenuation mechanism that acts at three separate sites within the operon, which are located in the 5Ј-untranslated leader, between the first (pyrR) and second (pyrP) genes, and between the second and third (pyrB) genes of the operon (3, 5). PyrR is proposed to regulate the ratio of terminated to readthrough transcripts at each attenuation site by permitting the formation of a -independent transcription terminator when exogenous pyrimidines are available. The binding of PyrR to pyr mRNA interferes with the formation of an alternative upstream stem-loop structure, the antiterminator, which is otherwise kinetically and thermodynamically favored. The presence of a conserved sequence within the 5Ј-stem of each antiterminator suggested a site within the pyr mRNA for interaction with PyrR (3); this site is the locus of several cis-acting mutants in the first pyr attenuator which are deficient in repression by pyrimidines (6).In addition, PyrR functions as a novel uracil phosphoribosyltransferase (UPRTase), 1 catalyzing the f...

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Section: Fig 1 Esi-ms Analysis Of Purified Pyrrmentioning

confidence: 76%

Purification and Characterization of Bacillus subtilis PyrR, a Bifunctional pyr mRNA-binding Attenuation Protein/Uracil Phosphoribosyltransferase

Turner¹,

Bonner²,

Grabner

et al. 1998

Journal of Biological Chemistry

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“…The Asp-Pro dipeptide appears to be universal among uracil phosphoribosyltransferases, as it is present in all the enzymes from the organisms mentioned above as well as yeasts (242), bacilli (243,244), and lactococci (245). Analysis of a mutant variant that had this particular proline residue replaced by an aspartate revealed a 100-fold increase in the K m value for uracil and a 50-fold reduction in the k cat value.…”

Section: Reactions At the Anomeric Carbon Of Prppmentioning

confidence: 99%

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

Hove‐Jensen

Andersen

Kilstrup

et al. 2017

Microbiol Mol Biol Rev

169

187

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SUMMARY 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.

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“…The three proteins show a high level of identity to each other, with the C. albicans protein being 72 and 56% identical to S. cerevisiae and T. gondii UPRT, respectively. The crystal structures of UPRT from two bacteria (Thermotoga maritima [structure is available from the Protein Data Bank at http://www.rcsb.org] and Bacillus caldolyticus [6]) and T. gondii (23) were reviewed, which revealed similarities in their protein folds as well as the fact that these enzymes tend to exist in various oligomeric forms. Analysis of the three-dimensional structure of UPRT from T. gondii, the organism bearing the closest phylogenetic relationship to C. albicans for which the crystal structure has been determined, reveals that Arg126 (cognate of Arg101 in C. albicans [ Fig.…”

Section: Codon (A T-to-c Transition)mentioning

confidence: 99%

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

Hope¹,

Tabernero

Denning³

et al. 2004

Antimicrob Agents Chemother

148

126

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Primary resistance in Candida albicans to flucytosine (5-FC) was investigated in 25 strains by identifying and sequencing the genes FCA1, FUR1, FCY21, and FCY22, which code for cytosine deaminase, uracil phosphoribosyltransferase (UPRT), and two purine-cytosine permeases, respectively. These proteins are involved in pyrimidine salvage and 5-FC metabolism. An association between a polymorphic nucleotide and resistance to 5-FC was found within FUR1 where the substitution of cytidylate for thymidylate at nucleotide position 301 results in the replacement of arginine with cysteine at amino acid position 101 in UPRT. Isolates that are homozygous for this mutation display increased levels of resistance to 5-FC, whereas heterozygous isolates have reduced susceptibility. Three-dimensional protein modeling of UPRT suggests that the Arg101Cys mutation disturbs the quaternary structure of the enzyme, which is postulated to compromise optimal enzyme activity. A single resistant isolate, lacking the above polymorphism in FUR1, has a homozygous polymorphism in FCA1 that results in a glycine-to-aspartate substitution at position 28 in cytosine deaminase.Flucytosine (5-FC) was initially synthesized in 1957 as an anticancer drug. Unlike 5-fluorouracil (5-FU), a closely related fluorinated pyrimidine, 5-FC did not exhibit antineoplastic activity but was subsequently found to possess antifungal activity and was used in 1968 to treat human cryptococcosis and candidiasis (25). Flucytosine administered in combination with amphotericin B remains the standard of care for cryptococcal meningitis, and the drug continues to have a role in the treatment of Candida infections which are life threatening or in circumstances where drug penetration may be problematic, such as infections of urine, eyes, and heart valves.Flucytosine is metabolized via the pyrimidine salvage pathway ( Fig. 1), where it acts as a subversive substrate with the subsequent production of toxic nucleotides and disruption of DNA and protein synthesis (18,26). After being actively transported into the cell by membrane permeases, 5-FC is converted via 5-FU to 5-fluoro-uridylate (synonymous with 5-fluoro-UMP ) under the action of the enzymes cytosine deaminase and uracil phosphoribosyltransferase (UPRT), respectively. 5-FUMP is in turn phosphorylated by two specific kinases to 5-fluoro-UTP, which is incorporated into RNA. 5-FUMP is also reduced to 5-fluoro-2Ј-deoxyuridylate, which inhibits the enzyme thymidylate synthetase and thus DNA synthesis by decreasing the available nucleotide pool. Mammalian cells lack the enzyme cytosine deaminase and consequently are not directly subject to the toxic effects of 5-FC.Primary resistance in Candida albicans, the focus of this paper, refers to inherent 5-FC resistance in the absence of prior drug exposure; in recent surveys, this resistance is observed in around 3% of isolates (1, 16). This phenomenon was recognized many years ago to be disproportionately represented in isolates belonging to serogroup B and more recently was found to ...

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Recombinant Uracil Phosphoribosyltransferase from the ThermophileBacillus caldolyticus:Expression, Purification, and Partial Characterization

Cited by 21 publications

References 24 publications

Purification and Characterization of Bacillus subtilis PyrR, a Bifunctional pyr mRNA-binding Attenuation Protein/Uracil Phosphoribosyltransferase

Purification and Characterization of Bacillus subtilis PyrR, a Bifunctional pyr mRNA-binding Attenuation Protein/Uracil Phosphoribosyltransferase

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

Molecular Mechanisms of Primary Resistance to Flucytosine in Candida albicans

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