INTRODUCTION

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In Salmonella enterica serovar Typhimurium LT2, purines and thiamine are synthesized via a divergent pathway where 5-aminoimidazole ribonucleotide (AIR) is the intermediate at the branch point (Fig. 1). Since the cellular purine requirement is approximately 10³-fold higher than the thiamine requirement (based on auxotrophic requirements), this pathway provides a model to address control of an important metabolic branch point. Previous genetic and molecular analyses demonstrated that even 1% of the wild-type level of AIR synthetase was sufficient to supply the cellular requirement for thiamine but not purines (J. L. Zilles and D. M. Downs, submitted for publication), indicating that thiamine synthesis can be maintained even when flux through the common pathway is severely reduced. Under this condition, thiamine synthesis could continue if levels of the substrate (presumed to be AIR) remained above the K_m for the first committed thiamine enzyme or if there were metabolite channeling between PurI and the thiamine enzyme (thought to be ThiC). Mutational analysis of purI, encoding the final common enzyme AIR synthetase, was pursued to clarify the parameters controlling thiamine synthesis with changing flux through the purine pathway.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Biosynthesis of purines and thiamine. Purine enzymes are noted below the arrow representing the reaction that they catalyze. THZ-P, 4-methyl-5-(-hydroxyethyl)thiazole monophosphate; HMP-PP, 4-amino-5-hydroxyethyl-2-methylpyrimidine pyrophosphate.

The synthesis of AIR, via the pathway common to purine and thiamine synthesis, appears to be regulated only in response to exogenous purines. There are three known levels of regulation on this pathway: (i) transcription of pur genes is repressed by PurR (with its corepressors hypoxanthine and guanine) (17, 18, 22, 28, 33, 39), (ii) allosteric inhibition of the first committed step in purine biosynthesis (phosphoribosylpyrophosphate amidotransferase, PurF) by AMP and GMP (24), and (iii) control of the levels of phosphoribosylpyrophosphate (PRPP), a substrate for the PurF enzyme. The level of PRPP in the cell drops substantially in the presence of exogenous purines (2, 19). Labeling studies suggest that exogenous adenine reduces flux through the purine biosynthetic pathway to 10% of that on minimal medium (32).

The current data on the purine-thiamine branch point are consistent with a model in which the flux to each branch of the purine-thiamine pathway depends on the concentration of AIR and the kinetic properties of the enzymes competing for AIR as a substrate. The primary phenotypic consequence of reduced flux through the common pathway is a purine requirement (Zilles and Downs, submitted). However, mutations that result in a thiamine (but not purine) requirement when flux through the purine pathway is reduced have been isolated and characterized (4, 5, 13, 30, 31). In general, these mutations appear to indirectly affect the thiamine biosynthetic pathway subsequent to the purine-thiamine branch point. The identification of mutations in the biosynthetic gene purI (encodes AIR synthetase in S. enterica, homologous to purM in Escherichia coli) that caused a conditional requirement for thiamine was unexpected. One of these mutants is the subject of this paper.

AIR synthetase catalyzes the conversion of formylglycinamidine ribonucleotide (FGAM) to AIR, ADP, and P_i (39) and has been purified and characterized from both chicken liver and E. coli (34, 35). Kinetic studies with the E. coli enzyme suggested a sequential mechanism in which ATP bound first and ADP was released last (35). The structure of AIR synthetase from E. coli has recently been solved, and the enzyme is believed to represent a new class of ATP-binding proteins (21, 27). The ATP-binding site in AIR synthetase was identified based on sequence alignments, structural considerations, and studies with an ATP affinity label (27). In this report we present the isolation of one purI mutant that can support purine synthesis but requires thiamine under some growth conditions. Biochemical analysis of the mutant PurI protein identified a defect in ATP binding that, in combination with the sequence analysis, supported the proposed location for the ATP-binding site of AIR synthetase (21, 27). Phenotypic and suppressor analyses indicated that high levels of FGAM were required for function of the mutant enzyme in vivo, suggesting that increased levels of FGAM can compensate for the decreased affinity of the mutant enzyme for ATP.

	MATERIALS AND METHODS

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General procedures. All strains used in this study are derivatives of S. enterica LT2 and are listed with their genotypes in Table 1. Unless otherwise indicated, strains were part of the lab collection or were constructed during the course of this work. Transductions were performed as described previously (12). Plasmid pPurF was isolated from a plasmid library (pBR328) of S. enterica DNA by its ability to complement a purF deletion mutant. PCR analysis confirmed the presence of the purF gene.

Strain construction. (i) Thiamine-requiring purI mutants. The purI coding region was amplified using the error-prone Vent (exo) polymerase. PCR conditions were as follows: initial denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1.5 min. Resulting PCR products were purified and blunt-end ligated into SmaI-cut pSU19, and the plasmids were moved into a purI null mutant (DM42) by electroporation as described (14), selecting for pSU19-encoded chloramphenicol resistance. The resulting colonies were screened for a purine-sensitive phenotype: growth on minimal medium and minimal medium supplemented with adenine and thiamine but no growth with adenine alone. Plasmids from putative purine-sensitive mutants were purified and reelectroporated to verify that the causative mutations were contained on the plasmid. To ensure independence, only one purine-sensitive mutant was saved from each amplification.

(ii) Site-directed mutagenesis. Site-directed mutagenesis was performed using the megaprimer method as described previously (3). The resulting PCR product was blunt-end ligated into SmaI-digested pSU19 and electroporated into a purI insertion mutant (DM42), selecting for chloramphenicol resistance, and replica printing to assess growth on minimal medium. Putative clones were confirmed by sequencing, and their phenotype was assessed by replica printing.

(iii) Generating chromosomal alleles of purI mutant. The plasmid-encoded purine-sensitive purI allele was removed from p103 by restriction digestion, gel purified, and ligated into the pMAK705 vector, which encodes a chloramphenicol resistance marker and has a temperature-sensitive origin of replication (16). The construct of interest was identified by electroporating into a purI null mutant (DM42) at 30°C (the permissive temperature), selecting for both chloramphenicol resistance and growth on minimal medium. A phage lysate was then grown on a strain containing the purI allele in pMAK705 and used to transduce a purI insertion mutant (DM2148) to prototrophy at 42°C (the restrictive temperature). Transductants were screened for chloramphenicol sensitivity, tetracycline sensitivity, and purine sensitivity, indicating loss of the plasmid, loss of the purI insertion, and presence of the mutant allele, respectively. The presence of the mutant allele was confirmed by sequence analysis.

(iv) Isolation and mapping of suppressor mutations. Six independent, spontaneous suppressor mutations were identified by plating saline cell suspensions of six independent overnight cultures of a strain carrying the purine-sensitive allele purI3098 (DM4794) on minimal glucose adenine and incubating at 37°C for 2 days. The suppressor phenotype was confirmed by replica printing and growth curves, and one suppressor mutant was saved from each culture (strains DM4829 to DM4834). To facilitate characterization of these spontaneous mutants, an insertion linked to a suppressor mutation was identified as follows. A suppressed strain (DM4829, purI3098 purR3105) was transduced to kanamycin resistance using phage grown on a MudJ pool. The transduction was performed on minimal medium to eliminate purine auxotrophs. Transductants were then screened for a purine-sensitive phenotype, indicating that the suppressor mutation had been replaced with a wild-type allele. The linkage between a MudJ insertion and the suppressor mutation was verified during reconstruction. The resulting insertion, zdx-9103::MudJ, was subsequently found to be linked to all six spontaneous suppressor mutations.

Biochemical analysis. (i) Protein purification. Plasmids were constructed containing the appropriate purI alleles in the pET20b vector, which added 13 amino acids to the carboxyl terminus of AIR synthetase (KLAAALEHHHHHH) (Novagen). The constructs were confirmed by phenotypic analysis for purine sensitivity in a purI null mutant of S. enterica and by sequence analysis before being electroporated into the E. coli strain BL21/(DE3) for overexpression. For unknown reasons, the plasmid containing the mutant allele was not stable in this background; induction of the R35C/E57G protein was therefore performed in the presence of an additional plasmid, pLysS (Novagen), which reduced preinduction expression and allowed maintenance of the plasmid. Cultures (12 ml) grown overnight in Luria-Bertani (LB) with carbenicillin (LB-Cb) were used to inoculate 400 ml of LB-Cb. The resulting culture was grown at 37°C with shaking to 80 Klett units (red filter), at which point solid IPTG (Fisher Biotech) was added to a final concentration of 0.4 mM. After 2 h of additional growth, the cultures were collected by centrifugation at 4°C at 5,000 × g for 10 min. Cell extracts were prepared as follows. Cell pellets were resuspended in 4 ml of buffer (500 mM NaCl, 20 mM Tris-HCl [pH 7.9], 5 mM imidazole), sonicated on ice, spun at 4°C at 39,000 × g for 20 min to remove debris, and filtered through a 0.45-µm filter (Gellman Laboratory).

(ii) Assays. AIR synthetase activity was assayed by detecting formation of AIR using a previously described modification of the Bratton-Marshall assay for diazotizable amines (34, 35). To conserve reagents, the assays were performed in small volumes. In a typical experiment, a volume of 40.5 µl contained 50 mM HEPES (pH 7.7), 20 mM MgCl₂, 150 mM KCl, and various concentrations of ATP and FGAM. The reaction mixture was equilibrated at 37°C, and the reaction was initiated by the addition of enzyme: 75 ng of wild-type AIR synthetase or 300 ng of mutant AIR synthetase in 4.5 µl. Enzymes were diluted in buffer containing 50 mM HEPES (pH 7.7), 50 mM KCl, and 10% glycerol. After 2 min, the reaction was stopped by the addition of 10 µl of 20% trichloroacetic acid-1.33 M potassium phosphate (pH 1.4). Remaining reagents (sodium nitrite [0.016% (wt/vol) final concentration], ammonium sulfamate [0.08% final concentration], N-(1-naphthyl)ethylenediamine dihydrochloride [0.01% final concentration]) were added as described to a final volume of 85 µl (34, 35). After allowing the color to develop for 30 min, absorbance was read at 500 nm in a quartz microtiter plate on the Spectramax Plus plate reader and corrected to a pathlength of 1 cm (Molecular Devices). For the determination of ATP kinetic constants, FGAM was constant at 100 µM and ATP was varied from 25 µM to 5 mM (wild-type enzyme) or 0.2 to 7.5 mM (mutant enzyme). For the determination of FGAM kinetic constants, ATP was constant at 0.5 mM (wild-type enzyme) or 5 mM (mutant enzyme), determined in an earlier experiment (data not shown) to be saturating (five times the K_m) for each enzyme, and FGAM was varied between 10 and 200 µM (wild-type enzyme) or 15 and 160 µM (mutant enzyme). At low substrate concentrations, one or more of the following was doubled: reaction time, amount of enzyme, or reaction volume. Control experiments showed the expected linear dependence of the amount of AIR produced on reaction time and amount of enzyme. Kinetic constants were calculated using the programs of Cleland (10) and plotted using Kaleidagraph (Abelbeck Software).

	RESULTS

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Isolation of purI mutations resulting in a conditional Thi phenotype. To address whether enzymes at the purine-thiamine branch point could alter the distribution of the common metabolite, purI was mutagenized. Strains in which the only source of AIR synthetase was a plasmid-encoded, PCR-mutagenized purI gene were grown on minimal medium and screened for thiamine auxotrophy in the presence of exogenous adenine. Two independent mutants were isolated and reconstructed, verifying the involvement of the plasmid-encoded purI allele in the phenotype. Plasmid p103 (strain DM3593) contained three mutations predicted to cause amino acid substitutions R35C, E57G, and R198W in AIR synthetase. The second plasmid, pTE1 (strain DM3995), also contained three mutations in the purI coding sequence, corresponding to amino acid changes of K31N, A50G, and L218R.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Purine-sensitive phenotype of R35C/E57G AIR synthetase mutant. Growth of the wild-type (A) and PurI R35C/E57G mutant (B) strains is shown. Inocula were grown to full density in minimal glucose medium before subculturing into minimal medium () or minimal medium with 0.4 mM adenine (). Solid symbols represent growth with 50 nM thiamine added. A representative experiment is shown; similar results were seen with at least four independent cultures.

R35C/E57G mutant AIR synthetase has an increased K_m for ATP. Thirteen amino acids (including six histidine residues) were fused to the carboxy terminus of wild-type and R35C/E57G mutant AIR synthetase, and the proteins were purified using a nickel affinity column. The purified fusion proteins were assayed for AIR synthetase activity as described previously (34, 35). Both the wild-type and mutant proteins were stable and active after purification. The kinetic plots are shown in Fig. 3, and the resulting kinetic constants are shown in Table 2. As anticipated from the high degree of sequence identity (92%), the kinetic constants of the wild-type S. enterica AIR synthetase were similar to those reported for the wild-type E. coli enzyme (35).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Kinetic analysis of purified S. enterica AIR synthetase. Data for the wild-type (open circles) and the R35C/E57G mutant (solid circles) strains are shown. (A) Concentration of ATP was fixed at approximately 5 Km, 0.5 mM (wild type) or 5 mM (mutant). (B) Concentration of FGAM was fixed at 0.1 mM and the ATP concentration was varied. The lines are least-squares fits of the kinetic data (solid, wild type; dotted, R35C/E57G mutant) to a hyperbolic rate expression, calculated using the programs of Cleland (10).

View larger version (47K): [in this window] [in a new window]   FIG. 4.   Location of mutated residues in S. enterica AIR synthetase. A model of S. enterica AIR synthetase was generated automatically by Swiss-Model (15) using the coordinates for E. coli AIR synthetase (PDB entry 1CLI) as input (21). As the two proteins are 92% identical (314 of 341 crystallographically located residues), it is anticipated that this is a highly accurate model. Each active site is defined by a groove composed of residues from both subunits in the dimer. The groove, which runs into the plane of the page, is bracketed by the ATP-binding loop (labeled ATP, at the front), initially identified by crystallography and affinity labeling (27), and an inorganic sulfate (at the back), proposed to locate the binding site for FGAM (21). The N termini of each subunit are labeled; in subunit A the first residue is Thr-5, and in subunit B the first residue is Ala-21. (Top) Locations of mutations in the R35C/E57G protein are shown in space-filling models. Note that R198W, found in strain DM3593, is not shown because it is unnecessary for the purine-sensitive phenotype. (Bottom) Locations of mutations in the K31N/A50G/L218R protein (strain DM3995) are shown in space-filling models. This figure was prepared using Molscript (20) and Raster3D (23).

Increased flux through the purine pathway suppresses the in vivo defect of R35C/E57G AIR synthetase. The biochemical parameters of the mutant enzyme were inconsistent with the initial model suggested by the in vivo phenotype, which predicted an increased K_m for FGAM. Exogenous purines have been reported to increase ATP levels slightly, which if anything might be expected to increase the activity of the mutant protein (2). However, in vivo the mutant protein appeared to be inhibited, either directly or indirectly, by exogenous purines. To resolve this paradox, we isolated and characterized the most frequent class of mutations that suppressed the purine-dependent thiamine requirement.

	DISCUSSION

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We report here the initial genetic and biochemical analyses of one phenotypic class of AIR synthetase mutants. Although the mutants were prototrophic on minimal medium, in the presence of exogenous purines they exhibited thiamine auxotrophy. This phenotype was unexpected, since previous analysis demonstrated that as little as 1% of the wild-type level of AIR synthetase was sufficient for growth in the presence of exogenous purines but not for growth on minimal medium (Zilles and Downs, submitted). The conditional thiamine requirement described here was suppressed by mutations that increased flux through the common pathway in the presence of purines. A simple interpretation of this genetic analysis suggested that the mutant enzyme might have an increased K_m for FGAM. However, kinetic analysis of the mutant AIR synthetase showed no change in the K_m for FGAM but a 13-fold increase in the K_m for MgATP.

While this work was in progress, new structural data on AIR synthetase became available. The AIR synthetase ATP-binding site was proposed on the basis of structural constraints and ATP analog affinity labeling (21, 27). Structurally, the mutant residues in this work (R35C and E57G) are near the proposed ATP binding site (Fig. 4), consistent with the mutations affecting the K_m for MgATP. Although a thorough analysis of the second reported purI mutant was not performed, it is worth noting that it also contained two amino acid substitutions near the proposed ATP binding site (Fig. 4). The mutant described in this work represents the first analysis of a mutant AIR synthetase enzyme with significant impairment of the ATP site. The structural analyses shown in Fig. 4 also show that the mutant residues are surface exposed and appear to lie in a single groove.

The connection between the high K_m for MgATP of the mutant enzyme and the resulting in vivo phenotype was not immediately apparent. It is likely that the nutritional requirement caused by the R35C/E57G mutant is a consequence of in vivo metabolite levels affecting the activity of the mutant PurI enzyme. Efforts to characterize these effects more thoroughly or to mimic them in vitro have been hampered by the difficulties involved in obtaining accurate measurements of metabolite pool sizes, particularly for unstable metabolites such as FGAM. Several possible models could explain the nutritional phenotype of the mutant in the context of biochemical parameters determined for this enzyme in vitro. These models include (i) increased FGAM altering the reaction order of the mutant enzyme, (ii) disruption of metabolite channeling in the mutant, (iii) stabilization of the mutant PurI by FGAM, and (iv) inhibition of the mutant PurI by elevated nucleotide levels.