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Originally published In Press as doi:10.1074/jbc.M405480200 on July 26, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40345-40350, September 24, 2004
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Heterooligomeric Phosphoribosyl Diphosphate Synthase of Saccharomyces cerevisiae

COMBINATORIAL EXPRESSION OF THE FIVE PRS GENES IN ESCHERICHIA COLI*

Bjarne Hove-Jensen{ddagger}

From the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-1307 Copenhagen, Denmark

Received for publication, May 17, 2004 , and in revised form, July 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({Delta}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 Pi, to be temperature-sensitive, and inhibited by ADP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The compound 5-phospho-D-ribosyl {alpha}-1-diphosphate (PRPP)1 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 [EC] ), which is encoded by a PRS gene: ribose 5-phosphate + ATP -> PRPP + 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 acids) and amino acid sequence identity (44–63%) (7). In contrast, S. cerevisiae PRPP synthase subunits 1 and 5 contain additional amino acids, which constitute non-homologous regions (NHRs). Thus, subunit 1 contains one NHR (NHR1) consisting of 105 amino acids, whereas subunit 5 contains NHR5-1, consisting of 110 amino acids, and NHR5-2, consisting of 63 amino acids. The "insertion points" of NHR1 and NHR5-2 are at similar locations, as seen by amino acid sequence alignments of PRS1- and PRS5-specified polypeptides. As a consequence of these NHRs, the lengths of subunits 1 and 5 are 427 and 496 amino acids, respectively. The amino acid sequence identity of subunit 1 or 5 with PRPP synthases from E. coli, B. subtilis, or human is 41–54%.

In general, microorganisms contain a single PRPP synthase-specifying gene. To elucidate the role of five PRS genes in S. cerevisiae, I have undertaken a biochemical and genetic analysis of the oligomerization of PRPP synthase by expression of S. cerevisiae PRS genes in E. coli. Genes encoding S. cerevisiae PRPP synthase subunits are designated by PRS in capital letters, whereas the E. coli PRPP synthase-encoding gene is designated prs. For clarity, the S. cerevisiae PRS genes may be designated as PRSSc and that of E. coli may be designated as prsEc.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Microbial Strains and Growth Media—The S. cerevisiae and E. coli K-12 strains used are listed in Table I. S. cerevisiae strain YN94-2 was grown at 30 °C in YPD medium (8) with aeration by shaking. E. coli was grown at 30 °C in NZY medium (9) or in AB minimal medium (10) with glucose (0.2%) as the carbon source. When necessary, cells were supplemented with guanosine (30 mg liter–1), uridine (20 mg liter–1), histidine (40 mg liter–1), tryptophan (40 mg liter–1), thiamin (1.0 mg liter–1), NAD (40 mg liter–1), {delta}-aminolevulinate (40 mg liter–1), kanamycin (30 mg liter–1), ampicillin (25 or 100 mg liter–1), or tetracycline (10 mg liter–1). DNA Technology—Methods for the isolation of plasmid DNA (11) and chromosomal DNA (12) from E. coli as well as for transformation of E. coli (13) have been described previously. Restriction and ligation of DNA were performed as described by the suppliers of restriction endonucleases (Amersham Biosciences, Promega, Roche Applied Science, and New England Biolabs) and T4 DNA ligase (Amersham Biosciences).


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  		TABLE I
Microbial strains and plasmids

The plasmid pBR322 was used as parent for recombinant plasmids. Each of the plasmids contained a bla gene. In addition, pBR322 contains a tetracycline resistance-encoding gene. Plasmid-harbored PRS genes were expressed from the E. coli prs promoter.

 
DNA of S. cerevisiae strain X2180-1A was used as template for the isolation of PRS genes by PCR. PRS1 was isolated with the oligodeoxyribonucleotides Y1u (5'-GACGCATGCCGGAGGTTCTTCTCATGCGTAAGTGTAAAATTTTTGTTGGTAACTC-3') and Y1d (5'-CTTAGATTCGAATTATTACAGAGCTGCCAAAGAGTCAAATAAAACGG-3'), PRS2 was isolated with PRS2-u (5'-CAAGACGCATGCCGGAGGTTCTTCTCATGTCTACAAACAG-3') and PRS2-d (5'-CTTAGATTCGAATTATTAAACAGGCGCATGTG-3'), PRS3 was isolated with P3u (5'-CAAGACGCATGCCGGAGGTTCTTCTCATGCCAACAAATTCCATC-3') and P3d (5'-ACTTAGATCGATTATTATAAGGGATAATTCTTAAATAAATAAG), PRS4 was isolated with P4-u (5'-CAAGACGCATGCCGGAGGTTCTTCTCATGGCTATAAATAGTATC-3') and P4-d (5'-CTTAGATTCGAATTACTATACTGGAGCATGGG-3'), and PRS5 was isolated with Y5u (5'-GACGCATGCCGGAGGTTCTTCTCATGTCAATGAGTAATATTGTTGTTTTTGGAGGGG-3') and Y5d (5'-CTTAGATTCGAATTACTACCATCCATGCTCGAACAACATAGAAATGG-3'). PCR products containing PRS1 or PRS5 were digested by restriction endonucleases SphI and BstBI and ligated to similarly restricted DNA of pHO11, which contains a wild-type version of the prsEc coding sequence together with flanking E. coli DNA sequences (387 and 301 bp upstream and downstream, respectively). PCR products containing S. cerevisiae PRS2, PRS3, or PRS4 were digested by restriction endonucleases SphI and BstBI and ligated to similarly restricted DNA of pHO4, which contains a wild-type version of the prsEc coding sequence together with flanking E. coli DNA sequences (514 and 547 bp upstream and downstream, respectively). Plasmid pHO4 was constructed by digesting DNA of pHO2 by restriction endonuclease AvaI followed by ligation. The effect of these procedures, which resulted in pHO405 (PRS1), pHO485 (PRS2), pHO491 (PRS3), pHO482 (PRS4), and pHO420 (PRS5), was replacement of the prsEc coding sequence with those of PRSSc and retaining the flanking sequences of E. coli origin to facilitate gene replacement in this organism.

Plasmids harboring tandem arrangements of PRSSc genes were constructed by PCR amplification of S. cerevisiae PRS2, PRS4, or PRS5 DNA with oligodeoxyribonucleotides that resulted in recognition sites for restriction endonuclease BstBI at both ends of the resulting DNA fragments. The DNA fragments were digested by BstBI and ligated to BstBI-digested DNA of plasmids harboring S. cerevisiae PRS1, PRS2, or PRS5, each of which had a BstBI recognition site located immediately downstream of the coding sequence. PRS5 was amplified with YP5Up2 (5'-CCACGATTCGAAGGAGTGCACGTATGTCAATGAGTAATATTGTTGTTTTTGG-3') and Y5D and with DNA of pHO420 as template. The resulting DNA fragment was ligated to DNA of pHO405 to obtain pHO443 (PRS1 PRS5) or ligated to DNA of pHO485 to obtain pHO503 (PRS2 PRS5). PRS2 was amplified with the oligodeoxyribonucleotides GP2U-3 (5'-GGCTACTTCGAAAGGAAACAGAATTCATGTCTACAAACAGTATTAAGTTG-3') and 2d (5'-CAGAAGCTTTTCGAAAGGCCTGCCGCGCGATGACCATGGTACGTACTCCTTATTAAACAGGCGCATGTG-3') and PRS2 DNA as template. The resulting DNA fragment was ligated to DNA of pHO405 to obtain pHO448 (PRS1 PRS2) or ligated to DNA of pHO482 to obtain pHO504 (PRS4 PRS2). PRS4 was amplified with GP4U (5'-CTAGTTCGAAGAGGAGAAATTCGATGGTCATCGACCTTGAGC-3') and GP4D (5'-GAATGGTTCGAATTACTATACTGGAGCATGGG-3') and PRS4 as the template. The resulting DNA fragment was ligated to DNA of pHO405 to obtain pHO438 (PRS1 PRS4) or ligated to DNA of pHO420 to obtain pHO429 (PRS5 PRS4).

The inserts of all the plasmids constructed with PCR-amplified DNA fragments were sequenced. The deduced amino acid sequences of S. cerevisiae PRS1, PRS2, PRS3, and PRS5 were identical to those published previously, whereas the nucleotide sequence of the PRS4 gene used in the present work differed slightly from that published previously (4, 5).2 Nucleotide sequencing was performed with an Applied Biosystems Model 310 genetic analyzer using the cycle sequencing method with dye terminators as recommended by the supplier (ABI Prism BigDye terminator cycle sequencing ready reaction kit, PE Applied Biosystems). The oligodeoxyribonucleotides used as primers were provided by Hobolth DNA Syntese (Hillerød, Denmark). PCR was performed in a Trio-Thermoblock (Biometra) with chromosomal or plasmid DNA as template by standard procedures using DNA polymerase from Pyrococcus furiosum (Invitrogen).

Complementation—PRPP-less strains ({Delta}prs) of E. coli, such as HO773, require purines and pyrimidines as well as histidine, tryptophan, and NAD (3). Complementation of the {Delta}prs allele or of a PRSSc gene by various plasmid-borne genes was analyzed by plating cells on minimal medium containing uridine, histidine, and tryptophan and with or without NAD or guanosine present. The presence of active PRPP synthase relieves one or more of these requirements dependent of the activity of the enzyme, i.e. the amount of PRPP produced. NZY medium contains all of these compounds but NAD. Thus, strain HO773 will grow in NZY medium only when supplemented with NAD. On the other hand, the formation of PRPP synthase activity in HO773 results in NAD prototrophy.

Gene Replacement—PRS genes were inserted into the E. coli chromosome in place of prsEc by using a two-step procedure involving a polA1 strain (HO480) and a pol+ {Delta}prs-4::Kanr hemA strain (HO2120) essentially as described before (14). Insertion of the various PRS genes at the proper position was confirmed by genetic mapping and by nucleotide sequencing of DNA fragments produced by PCR. The template for this PCR was chromosomal DNA isolated from each strain. Primers annealed outside the replaced DNA fragment. In all of the strains used, the predicted gene replacement had occurred. Bacteriophage P1 transduction was performed as described previously (15).

Preparation of Cell Extracts for Enzyme Analysis—One hundred milliliters of S. cerevisiae culture grown to saturation in YPD medium was harvested by centrifugation, resuspended in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.6) containing phenylmethylsulfonyl fluoride (1 mM), EDTA (1 mM), EGTA (1 mM), leupeptin (Sigma) (1 mg liter–1), and mixed with 1 ml of glass beads (~0.5 µm) (Sigma). Cells were broken by agitation in a Mikro-Dismembrator U (Braun Biotech International) at a frequency of 1600 s–1 for 2.5 min (16). Cell debris was removed by centrifugation to form a crude extract. E. coli was grown to saturation in NZY medium supplemented with appropriate antibiotics. Cells were harvested by centrifugation, resuspended in 50 mM potassium phosphate buffer (pH 7.6), and broken by ultrasonic treatment. The debris was removed by centrifugation. When the indicated ammonium sulfate was added to a crude extract, the precipitate was collected by centrifugation, dissolved in 50 mM potassium phosphate buffer, pH 7.6, and dialyzed against the same buffer.

Assay of PRPP Synthase Activity—PRPP synthase activity was assayed as follows unless otherwise stated. At zero time, 10 µl of extract, appropriately diluted and prewarmed at 30 °C, was mixed with 90 µlof a reaction mixture, prewarmed at 30 °C, to give the following final concentrations: 50 mM potassium phosphate buffer (pH 8.5), 5 mM ribose 5-phosphate, 4 mM magnesium chloride, 20 mM sodium fluoride, 15 mM phosphoenolpyruvate (Roche Applied Science), 1 µmol min–1 of pyruvate kinase (Roche Applied Science), 2 mM (10 GBq mol–1) [{gamma}-32P]ATP, prepared as described previously (17). Samples (10 µl) were removed after 1, 3, and 6 min of incubation at 30 °C and mixed with 5 µl of 0.33 M formic acid. This 15 µl was applied to a polyethyleneimine-coated cellulose thin-layer chromatography plate, prepared as described previously (18). The chromatogram was dried and developed for 18 cm in 0.85 M potassium phosphate, which had been titrated to pH 3.4 with 0.85 M phosphoric acid, to separate radiolabeled ATP and PRPP, which were quantitated in an Instant Imager (Packard Instrument Co.). Ribose 5-phosphate-dependent formation of PRPP was determined by parallel incubations as above but without ribose 5-phosphate present. Activity without Pi present was determined as above except that the potassium phosphate buffer was replaced by Tris-HCl buffer (pH 8.5). Protein content was determined by the bicinchoninic acid procedure with bovine serum albumin as the standard (19).

Determination of PRPP and Ribonucleoside Triphosphate Pools— E. coli cells were grown at 30 °C in Tris-buffered minimal medium containing 0.3 mM Pi (17) with glucose as the carbon source and with guanosine, uridine, histidine, and tryptophan present. After several generations of exponential growth, carrier-free 32Pi (Nex-053, PerkinElmer Life Sciences) was added to a specific radioactivity of ~3 TBq mol–1. After three generations of growth, samples were removed, and PRPP and ribonucleoside triphosphates were extracted and separated by two-dimensional thin-layer chromatography on polyethyleneimine-impregnated cellulose plastic sheets (17). Radioactivity was quantitated in an Instant Imager (Packard Instrument Co.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of and Complementation by PRS Genes—The five PRS genes were cloned in pHO4 or pHO11, which are derivatives of pBR322, in a manner that caused replacement of the prsEc coding sequence with those of the five PRS genes. There was a 3-fold advantage with these constructions. Firstly, it allowed expression of the PRS genes in E. coli to be driven by the well characterized prsEc promoter. Secondly, the presence of E. coli prs-flanking nucleotide sequences in the recombinant PRS-harboring plasmids allowed gene replacement by homologous recombination to occur, and a series of isogenic strains was obtained. These strains differed only in their PRS coding sequences. Finally, with all of the five PRS genes present in either the chromosome of E. coli or a plasmid, the effect of combining any two PRS genes could be analyzed. Complementation was performed as described under "Experimental Procedures." Briefly PRPP-less mutants of E. coli require guanosine, uridine, histidine, tryptophan, and NAD for growth. Transformants were plated on medium lacking guanosine or NAD, and growth in the absence of either of these compounds indicates acquisition of a PRS gene that specifies active PRPP synthase. Complementation was analyzed in the following strains: HO773, which contains a large deletion of the prsEc gene, resulting in lack of PRPP synthase activity; HO2264, HO2268, HO2480, HO2284, and HO2481, which had the prsEc gene replaced by the S. cerevisiae PRS1, PRS5, PRS2, PRS3, and PRS4 gene, respectively. These six strains required NAD for growth, indicating the formation of inactive PRS gene products. In addition, none of the five PRS genes (harbored in pHO405, pHO485, pHO491, pHO482, or pHO420) were able to complement the {Delta}prs mutant allele harbored in strain HO773, as seen by the requirement of NAD for the growth of the transformants (Table II). In contrast, a plasmid-borne wild-type allele of E. coli prs complemented {Delta}prs. As expected, the non-prs harboring plasmid pBR322 was unable to complement {Delta}prs. Strain HO2264 (PRS1) transformed with the PRS3-harboring plasmid pHO491 was able to grow in the absence of guanosine, which suggests that the PRS1-specified polypeptide together with the PRS3-specified polypeptide resulted in the formation of active PRPP synthase. If a less severe selective pressure was applied by omitting NAD rather than guanosine, the growth of HO2264 transformed with pHO485 (PRS2) was observed, which indicated the formation of an enzyme with low activity. PRS1 (pHO405), PRS4 (pHO482), or PRS5 (pHO420) did not complement PRS1. Strain HO2268 (PRS5) grew without guanosine present when transformed with PRS2-harboring or PRS4-harboring plasmids (pHO485 and pHO482, respectively) but required guanosine and NAD when transformed with plasmids harboring S. cerevisiae PRS1, PRS3, or PRS5 (pHO405, pHO491, and pHO420, respectively). Thus, PRS5-specified polypeptide together with polypeptide specified by PRS2 or PRS4 appeared to result in the formation of active PRPP synthase. The PRS2 gene (strain HO2480) was complemented by PRS5 (pHO420), and when the less selective pressure without NAD present was applied, also by PRS1 (pHO405). PRS3 (strain HO2284) was complemented by PRS1 (pHO405). Finally, PRS4 (strain HO2481) was complemented by PRS5 (pHO420) when guanosine was omitted and by PRS1 (pHO405) when NAD was omitted, indicating the formation of active PRPP synthase by subunit 4 together with subunit 5, and to a lesser extent, subunit 4 with subunit 1. The data of Table II show that identical results, with one exception, were obtained in reciprocal complementation, i.e. the same result was obtained with one gene in the chromosome and another in a plasmid and vice versa. The exception was PRS1 and PRS4, which only resulted in active enzyme with PRS1 expressed from a plasmid. Each strain responded identically when transformed with pHO11 or when transformed with pBR322. Although I did not directly analyze expression of the five plasmid-harbored PRS genes, the results described above show that each of them were indeed expressed as at least one pairwise combination involving each of the five genes produced active PRPP synthase.


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  		TABLE II
Complementation of {Delta}prs and PRSSc by PRSSc genes

Cells were plated on minimal medium supplemented with uridine, histidine, tryptophan, and NAD. Growth was recorded after 48 h of incubation at 30 °C: –, no growth; +, growth. Growth of all of the transformants with guanosine present as well as uridine, histidine, tryptophan, and NAD: +. Data in parenthesis show growth in the presence of guanosine, uridine, histidine, and tryptophan (i.e. in the absence of NAD) when the response was different from that recorded in the absence of guanosine.

 
I furthermore constructed plasmids harboring artificial bicistronic operons. Of these constructions, those harboring S. cerevisiae PRS1 PRS2, PRS5 PRS2, or PRS5 PRS4 complemented {Delta}prs, resulting in an NAD+ Guo+ phenotype, whereas that harboring PRS1 PRS4 resulted in a NAD+ Guo– phenotype. The constructions with S. cerevisiae PRS1 PRS5, PRS2 PRS3, PRS2 PRS4, PRS3 PRS4, or PRS3 PRS5 did not complement {Delta}prs (data not shown), i.e. a result similar to that described above with pairwise combinations of chromosomal and plasmid-borne genes. A plasmid harboring S. cerevisiae PRS1 PRS3 was not constructed.

PRPP Synthase Activity, PRPP, and Ribonucleoside Triphosphate Pools of E. coli Strains Harboring Two PRS Genes— Extracts of the strains that contained two PRS genes with a resulting NAD+ Guo– or NAD+ Guo+ phenotype were analyzed for their content of PRPP synthase activity (Table III). The data show that the activity of strain HO2284 (PRS3)/pHO405 (PRS1) was 350 nmol (min x mg of protein)–1, whereas the remaining strains (i.e. those containing S. cerevisiae PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, PRS5 PRS4) had no detectable PRPP synthase activity. Similarly, extract of strain HO2264 (PRS1)/pHO491 (PRS3) contained no detectable activity. For comparison, values for PRPP synthase activity determined in extracts of defined wild-type strains of S. cerevisiae and E. coli are also given in Table III. This lack of PRPP synthase in extracts of most of the strains may seem surprising as most of them grew in the absence of any addition, and thus, should contain readily detectable amounts of PRPP synthase activity. I therefore analyzed the synthesis of PRPP in vivo by determination of PRPP pools as well as of ribonucleoside triphosphate pools (Table III). Apart from strain HO2281 (PRS4)/pHO405 (PRS1), which was unable to grow in the low phosphate medium used for pool analysis, the strains produced detectable amounts of PRPP, albeit in very different amounts. Consistent with their NAD+ Guo– phenotype, the strains harboring S. cerevisiae PRS1 PRS2 contained very low PRPP pools, whereas their ribonucleoside triphosphate pools were essentially normal when compared with the pools of the E. coli wild-type strain HO698 (Table III). The remaining strains analyzed revealed increased pool sizes of PRPP as well as of ribonucleoside triphosphates, which may indicate altered regulation of the synthesis of these compounds.


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  		TABLE III
PRPP synthase activity, PRPP, and ribonucleoside triphosphate pools in E. coli strains harboring pairwise S. cerevisiae PRS genes with {Delta}prs-complementing activity (NAD+-phenotype)

All of the strains were NAD-prototrophic. Their phenotype with respect to guanosine requirement is indicated (cf. Table II). Guo+, guanosine prototrophic; Guo–, guanosine auxotrophic. ND, not determined. The data for E. coli strain HO698 have been published previously (3).

 
E. coli Strains Harboring Three PRS Genes—Certain combinations of three PRS genes had remarkable effects on the measurable PRPP synthase activity. The results are given in Table IV. None of the plasmids harboring two PRS genes apparently specified measurable PRPP synthase activity. However, introduction of the PRS1 PRS5-specifying plasmid, pHO443, into a PRS2 or a PRS4 background resulted in a large stimulation in PRPP synthase activity in vitro. The level was undetectable in strains HO2480 or HO2481 but 230 nmol (min x mg protein)–1 in strain HO2480/pHO443 and 150 nmol (min x mg protein)–1 in strain HO2481/pHO443. Plasmid pHO443 alone did not specify active PRPP synthase. Also, the PRS4 PRS5-specifying plasmid, pHO429, greatly stimulated the in vitro activity of a PRS1 (strain HO2264) or PRS2 (HO2280) background, again from an undetectable level to 62 or 170 nmol (min x mg protein)–1, respectively. This stimulatory effect was not general. The PRS1 PRS2-specifying plasmid, pHO448, increased PRPP synthase activity only in the PRS5 background (HO2268) from undetectable to 100 nmol (min x mg protein)–1, whereas in a PRS4 background (HO2481), no activity was detectable with or without pHO448. In the PRS4 background, however, a large stimulation in PRPP synthase activity was observed by introduction of the PRS2 PRS5-specifying plasmid, pHO503 (undetectable level versus 110 nmol (min x mg protein)–1). Finally, there was no stimulation in PRPP synthase activity by introducing pHO439 (PRS1 PRS4) into the PRS2 background (HO2480). In summary, a large activity was seen by combining subunits 1, 2, and 5, by combining subunits 1, 4, and 5, or by combining subunits 2, 4, and 5. In addition, extracts containing subunits 1 and 3 in combination with subunit 2, 4, or 5 contained detectable PRPP synthase activity, although the activity of the enzyme consisting of subunits 1, 3, and 5 was substantially lower than that of the enzymes consisting of subunits 1, 2, and 3 or 1, 3, and 4. In contrast, combining subunits 1, 2, and 4 did not result in detectable PRPP synthase activity, consistent with the NAD+ Guo– phenotype of these strains. Finally, combining PRS2 PRS3 PRS4 (strain HO2284 (PRS3)/pHO504 (PRS2 PRS4)) resulted in an NAD– Guo– phenotype and in undetectable PRPP synthase activity (Table IV).


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  		TABLE IV
PRPP synthase activity in E. coli strains harboring three PRS genes

 
Characterization of PRS-specified PRPP Synthase Synthesized in E. coli—The properties of PRPP synthases specified by S. cerevisiae PRS1 PRS3 and by S. cerevisiae PRS1 PRS2 PRS5 were analyzed in greater detail with respect to Pi, pH, and temperature dependence, as well as to inhibition by ADP. The results were compared with PRPP synthases of wild-type S. cerevisiae and E. coli (Table V). The two enzymes containing subunits 1 and 3 or subunits 1, 2, and 5 behaved quite similarly. Both required Pi for activity, they were essentially inactive at a low pH (6.0), they were inactive at a high temperature (56 °C), and both were severely inhibited by ADP. Wild-type S. cerevisiae PRPP synthase was also very sensitive to heat. In contrast, the effects of lack of Pi, low pH, and the presence of ADP on the wild-type enzyme was less severe than that on the recombinant enzymes.


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  		TABLE V
Properties of PRPP synthases containing subunits 1 and 3 or subunits 1, 2, and 5

PRPP synthase activity was determined in extracts of cultures grown to saturation in NZY medium (plasmid-containing E. coli strains) or in YPD medium (S. cerevisiae wild type strain YN94-2). The enzyme preparations of strains HO2284/pHO405 and HO2480/pHO443 were 30–60% ammonium sulfate fractions of crude extracts. PEP, phosphoenolpyruvate; PK, pyruvate kinase. Numbers in parenthesis indicate specific activity in nmol/(min x mg protein).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present work, I have shown for the first time that active PRPP synthase may be assembled from subunits, which are individually inactive. All well characterized PRPP synthases are homooligomeric enzymes with the exception of the rat liver enzyme, which is composed of two different but very similar polypeptides. Both of these polypeptides are active individually (20). I used an E. coli {Delta}prs strain with no endogenous PRPP synthase activity to detect the formation of active PRPP synthase specified by the five PRS genes. Individually, none of the five PRS genes specified active PRPP synthase as evaluated by the lack of growth of the {Delta}prs/PRSSc strains. The growth of {Delta}prs/PRSSc strains was not observed even in the absence of NAD. This is particularly striking as only 1–2% of the PRPP formed in wild-type E. coli cells are used for NAD synthesis (21). In contrast, each subunit together with at least one additional subunit was able to generate an active enzyme but not just any pairwise combination of subunits. Only 5 of the 10 possible two-subunit combinations appeared to have PRPP synthase activity. Previous analysis of S. cerevisiae revealed interaction in vivo between subunit 1 and 3, between subunit 5 and 2, and between subunit 5 and 4 (22). In the present work, I showed that co-expression of S. cerevisiae PRS1 and PRS3, PRS5 and PRS2, or PRS5 and PRS4 in E. coli resulted in the formation of active PRPP synthase. In addition, co-expression of S. cerevisiae PRS1 and PRS2 or PRS1 and PRS4 resulted in the formation of active PRPP synthase. These two latter combinations escaped detection in previous analysis of S. cerevisiae and demonstrate the power of using an E. coli {Delta}prs strain for this analysis. It is worth noting that the strains, which expressed PRS1 PRS2, had an NAD+ Guo– phenotype, and consistently, contained very little PRPP. It appears that the formation of active PRPP synthase required the presence of one subunit, which contained an NHR together with one subunit, which did not contain an NHR, i.e. subunit 1 together with one of either subunit 2, 3, or 4 or subunit 5 with subunit 2 or 4. In particular, combining subunits 2, 3, and 4 resulted in an inactive enzyme. This is consistent with results obtained by genetic studies, which showed that an S. cerevisiae {Delta}prs1 PRS2 PRS3 PRS4 {Delta}prs5 mutant strain was not viable (22). In a few cases, a gene dose effect was observed. Thus, expression of PRS1 and PRS3 resulted in dramatically different activities whether PRS1 was on the chromosome or on the plasmid, with PRS1 on the chromosome resulting in the higher activity. Apart from different expression levels, the codon bias may be of importance. The different codon bias of S. cerevisiae and E. coli (23) is also seen among all five PRS genes.

S. cerevisiae cells deleted for one, two, or three PRS genes contain very little PRPP synthase activity in vitro, from 0.9% (strain {Delta}prs2 {Delta}prs3) to 16–17% (strain {Delta}prs4, strain {Delta}prs5, or strain {Delta}prs2 {Delta}prs4 {Delta}prs5) of the activity of a wild-type strain (22). A {Delta}prs3 {Delta}prs4 strain contained 5.6% of the activity of a wild-type strain. I chose to analyze in further detail the PRPP synthases containing either subunits 1 and 3 or subunits 1, 2, and 5. These two enzymes should be comparable with those harbored in the S. cerevisiae mutant strains {Delta}prs2 {Delta}prs4 {Delta}prs5 and {Delta}prs3 {Delta}prs4, respectively. The two enzymes responded essentially similarly to the assay conditions applied here, i.e. lack of Pi, low pH, high temperature, and the presence of ADP, a potential inhibitor of PRPP synthases from enteric bacteria and from B. subtilis (24, 25). The wild-type S. cerevisiae PRPP synthase was less sensitive to ADP and retained a higher activity in the absence of Pi and a low pH than the recombinant enzymes. These differences may be ascribed to the incompleteness of the recombinant enzymes. Indeed, S. cerevisiae PRPP synthase appears to represent an example of a complex enzyme. Two different subunits are necessary to establish an active enzyme in vivo as confirmed by complementation of E. coli {Delta}prs as well as by the formation of PRPP in vivo by, for example, S. cerevisiae PRS2 PRS5. However, apart from PRS3 PRS1, no PRPP synthase activity could be detected in vitro unless a third PRS gene was co-expressed as well.


   FOOTNOTES
 
* This work was supported by the Danish Natural Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) X75075 [GenBank] (PRS2) and AJ245726 [GenBank] (PRS4) Back

{ddagger} To whom correspondence should be addressed: Dept. of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, 83H Sølvgade, DK-1307 Copenhagen K, Denmark. Tel.: 45-3532-2027; Fax: 45-3532-2040; E-mail: hove{at}mermaid.molbio.ku.dk.

1 The abbreviations used are: PRPP, 5-phospho-D-ribosyl {alpha}-1-diphosphate; NHR, non-homologous region. Back

2 The nucleotide sequence of the PRS4 gene used in the present work differed slightly from that published previously. Codon 82 specified lysine in the present sequence rather than glutamine, whereas codon 161 specified threonine in the present sequence rather than alanine, and the codon specifying arginine 162 in the original sequence was absent in the present. Back


   ACKNOWLEDGMENTS
 
I thank Michael Schweizer for providing S. cerevisiae strain YN94-2 and Steen Holmberg for providing S. cerevisiae DNA. Tonny D. Hansen is gratefully acknowledged for pertinent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hove-Jensen, B. (1988) J. Bacteriol. 170, 1148–1152[Abstract/Free Full Text]
  2. Khorana, H. G., Fernandes, J. F., and Kornberg, A. (1958) J. Biol. Chem. 230, 941–948[Free Full Text]
  3. Hove-Jensen, B. (1989) Mol. Microbiol. 3, 1487–1492[Medline] [Order article via Infotrieve]
  4. Carter, A. T., Narbad, A., Pearson, B. M., Beck, K. F., Logghe, M., Contreras, R., and Schweizer, M. (1994) Yeast 10, 1031–1044[CrossRef][Medline] [Order article via Infotrieve]
  5. Hernando, Y., Parr, A., and Schweizer, M. (1998) J. Bacteriol. 180, 6404–6407[Abstract/Free Full Text]
  6. Carter, A. T., Beiche, F., Hove-Jensen, B., Narbad, A., Barker, P. J., Schweizer, L. M., and Schweizer, M (1997) Mol. Gen. Genet. 254, 148–156[CrossRef][Medline] [Order article via Infotrieve]
  7. Krath, B. N., Eriksen, T. A., Poulsen, T. S., and Hove-Jensen, B. (1999) Biochim. Biophys. Acta 1430, 403–408[CrossRef][Medline] [Order article via Infotrieve]
  8. Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics, p. 207, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  9. Hove-Jensen, B., and Maigaard, M. (1993) J. Bacteriol. 175, 5628–5635[Abstract/Free Full Text]
  10. Clark, D. J., and Maaløe, O. (1967) J. Mol. Biol. 23, 99–112[CrossRef]
  11. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7, 1513–1523[Abstract/Free Full Text]
  12. Silhavy, T. J., Berman, M. L., and Enquist, L. W. (1984) Experiments with Gene Fusions, pp. 137–139, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  13. Mandel, M., and Higa, A. (1970) J. Mol. Biol. 53, 159–162[CrossRef][Medline] [Order article via Infotrieve]
  14. Bower, S. G., Harlow, K. W., Switzer, R. L., and Hove-Jensen, B. (1989) J. Biol. Chem. 264, 10287–10291[Abstract/Free Full Text]
  15. Miller, J. H. (1992) A Short Course in Bacterial Genetics, pp. 268–274, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Jazwinski, S. M. (1990) Methods Enzymol. 182, 154–174[Medline] [Order article via Infotrieve]
  17. Jensen, K. F., Houlberg, U., and Nygaard, P. (1979) Anal. Biochem. 98, 254–263[CrossRef][Medline] [Order article via Infotrieve]
  18. Hove-Jensen, B. (1992) J. Bacteriol. 174, 6852–6856[Abstract/Free Full Text]
  19. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76–85[CrossRef][Medline] [Order article via Infotrieve]
  20. Sonoda, T., Ishiharu, T., Ishijima, S., Kita, K., Ahmad, I., and Tatibana, M. (1998) Biochim. Biophys. Acta 1387, 32–40[CrossRef][Medline] [Order article via Infotrieve]
  21. Jensen, K. F. (1983) in Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms (Munch-Petersen, A., ed), pp. 1–25, Academic Press, London
  22. Hernando, Y., Carter, A. T., Parr, A., Hove-Jensen, B., and Schweizer, M. (1999) J. Biol. Chem. 274, 12480–12487[Abstract/Free Full Text]
  23. Nakamura, Y., Gojobori, T., and Ikemura, T. (2000) Nucleic Acids Res. 28, 292[Abstract/Free Full Text]
  24. Switzer, R. L., and Sogin, D. C. (1973) J. Biol. Chem. 248, 1063–1073[Abstract/Free Full Text]
  25. Arnvig, K., Hove-Jensen, B., and Switzer, R. L. (1990) Eur. J. Biochem. 192, 195–200[Medline] [Order article via Infotrieve]
  26. Hove-Jensen, B. (1983) J. Bacteriol. 154, 177–184[Abstract/Free Full Text]
  27. Sørensen, K. I., and Hove-Jensen, B. (1996) J. Bacteriol. 178, 1003–1011[Abstract/Free Full Text]
  28. Post, D., Switzer, R. L., and Hove-Jensen, B. (1996) Microbiology (Reading) 142, 359–365[Abstract/Free Full Text]
  29. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heynecker, H. L., Boyer, H. W., Crosa, J. H., and Falkow, S. (1977) Gene (Amst.) 2, 95–113[Medline] [Order article via Infotrieve]
  30. Hove-Jensen, B. (1985) Mol. Gen. Genet. 201, 269–276[CrossRef][Medline] [Order article via Infotrieve]

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