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Originally published In Press as doi:10.1074/jbc.M604429200 on September 20, 2006

J. Biol. Chem., Vol. 281, Issue 45, 33892-33899, November 10, 2006
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Inhibition of Fructose-1,6-bisphosphatase by Aminoimidazole Carboxamide Ribotide Prevents Growth of Salmonella enterica purH Mutants on Glycerol*

Michael J. Dougherty1, Jeffrey M. Boyd, and Diana M. Downs2

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, May 9, 2006 , and in revised form, September 20, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
An efficient cellular metabolism that can adapt to changing environmental conditions requires the integration of many biochemical pathways. Subtle interactions that exist between pathways in the metabolic network are in general not well understood. One level of these interactions involves metabolites common to multiple pathways. Cellular metabolites can impact physiology through roles in transcriptional regulation, translational regulation, or by mediating allosteric effects on key enzymes. One such central metabolite with regulatory capabilities is 5-amino-4-imidazole carboxamide ribotide (AICAR).3

AICAR is a purine nucleotide biosynthetic intermediate and the substrate for AICAR transformylase/IMP cyclohydrolase, encoded by the purH gene in Salmonella enterica. AICAR is also generated as a byproduct of histidine biosynthesis, released by the enzyme imidazole glycerol-phosphate synthase (Fig. 1A). AICAR has been shown to impact different metabolic pathways in diverse organisms. Bochner and Ames (1) proposed that 5-amino-4-imidazole carboxamide riboside 5'-triphosphate formed from the accumulation of AICAR is an alarmone for C1-folate deficiency in S. enterica, although this conclusion, in its simplest form, was not supported by subsequent work (2). AICAR has been shown to be a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli (3) and was recently shown to affect regulation of purine biosynthesis genes in Saccharomyces cerevisiae (4). Previous work from our laboratory demonstrated that AICAR accumulation inhibits the conversion of aminoimidazole ribotide to 4-amino-5-hydroxymethyl-2-methyl pyrimidine phosphate in the biosynthesis of thiamine pyrophosphate in S. enterica (5).

In mammalian systems, AICAR has been shown to activate AMP-activated protein kinase (6), which plays a critical role in energy balance and the response to metabolic stress (7). Through activation of AMP-activated protein kinase, AICAR inhibits proliferation of several types of cancer cells (8, 9). Furthermore, AICAR has been shown to inhibit gluconeogenesis in mammals due to inhibition of fructose-1,6-bisphosphatase (10, 11).

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11 [EC] ; FBP) catalyzes the hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P) to fructose 6-phosphate and inorganic phosphate (12). FBP is important for the regulation of flux between glycolysis and gluconeogenesis (Fig. 1B); as the enzyme is inhibited by AMP and fructose 2,6-bisphosphate (Fru-2,6-P) (13). Fru-2,6-P is a competitive inhibitor with respect to the substrate Fru-1,6-P, which binds at the active site (14, 15), whereas AMP binds to an allosteric site (16, 17), which causes a structural transition from the active R state of the enzyme to the inactive T state (18). FBP from mammalian sources has been extensively studied at the biochemical level. In Escherichia coli, FBP activity is required for growth on glycerol, succinate, or acetate as sole carbon and energy source (19). E. coli FBP has been shown to be activated by Mg2+ and inhibited by AMP and Fru-2,6-P, similar to the mammalian enzymes, although Mg2+ activation and AMP inhibition did not display cooperativity, as in the mammalian enzymes (20). The physiological relevance of Fru-2,6-P inhibition of E. coli FBP is not clear; Fru-2,6-P inhibition is not synergistic with AMP inhibition as in mammalian systems (20, 21), and to date Fru-2,6-P has not been found in bacterial cells (22). Phosphoenolpyruvate has been shown to relieve AMP inhibition of E. coli FBP (21, 23). Recently, the crystal structure of E. coli FBP was reported; these authors also demonstrated activation of FBP by phosphoenolpyruvate and postulated a novel allosteric binding site for this compound (24).


Figure 1
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  		FIGURE 1.
A, the purine and histidine biosynthetic pathways in S. enterica. B, glycolysis, gluconeogenesis, and the glycerol utilization pathways in S. enterica. Gene products catalyzing the reaction are indicated above the relevant steps.

 
The studies described here demonstrate that purH mutants of S. enterica are unable to use glycerol or other gluconeogenic carbon sources in the absence of exogenous histidine. Results show that this growth phenotype is due to inhibition of FBP by the accumulation of AICAR. AICAR-resistant alleles of fbp were isolated and the mutant proteins were purified and characterized in vitro. FBP is inhibited by AICAR at physiologically relevant concentrations and thus this interaction may represent a form of regulation relevant under some growth conditions, such as folate stress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains, Media, and Chemicals
All strains used in this study are derivatives of S. enterica LT2 and are listed with their genotypes in Table 1. The purH355 allele contains a thymidine inserted at position 716, resulting in a frameshift and generating a truncated PurH protein of 266 amino acids. MudJ refers to the Mud1734 insertion element (25) and Tn10d(Tc) refers to the transposition-defective mini-Tn10 described by Way et al. (26). No-carbon E medium supplemented with 1 mM MgSO4 (27, 28) and glucose (11 mM) or glycerol (22 mM) was used as a minimal medium. Difco nutrient broth (8 g/liters) with NaCl (5 g/liters) and Luria-Bertani broth were used as rich media. Difco BiTek agar was added (15 g/liter) for solid medium. When present in the media, supplements were provided at the following final concentrations: thiamine, 10 or 100 nM; adenine, 0.4 mM; histidine, 74 µM. When needed, antibiotics were added to the following concentrations in rich/minimal media: tetracycline, 20/10 µg/ml; kanamycin, 50/125 µg/ml; chloramphenicol, 20/4 µg/ml; and ampicillin, 50/15 µg/ml. Restriction enzymes and DNA ligase were purchased from Promega (Madison, WI). Cloned Pfu DNA polymerase was purchased from Stratagene (La Jolla, CA).


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  		TABLE 1
Strains and plasmids

All strains and plasmids were generated for this study or are part of laboratory stock unless indicated.

 
Genetic Methods
Phenotypic Analysis—Nutritional requirements were assessed on solid medium with soft agar overlays and by quantification of growth in liquid media. Protocols for each have been previously described (29, 30).

Transduction Methods—The high-frequency generalized transducing mutant version of bacteriophage P22 (HT105/1 int-201) was used in all transductions (31, 32). The methods for transduction and purification of transductants have been previously described (33).

Isolation of Linked Insertions—Transposons (Tn10d(Tc)) genetically linked to the mutation allowing utilization of glycerol as sole carbon source in a purH mutant were isolated by standard genetic techniques (34). The chromosomal location of each insertion was determined by sequencing using a PCR-based protocol (35, 36). A DNA product was amplified with degenerate primers and primers derived from the Tn10d(Tc) insertion sequence as described and sequenced at the University of Wisconsin Biotechnology DNA Sequence Facility.

Molecular Biology Techniques
The fbp gene was amplified from wild-type and mutant strains using genomic DNA as a template and the forward primer, 5'-TACTTTGCTGGCGCGATTCACCTG-3', and the reverse primer, 5'-TGTTTTCGTTTCCGCCTCATCTGG-3'. The resulting PCR products were purified and ligated into SmaI-cut pSU19. Plasmids were transformed into E. coli strain DH5{alpha} and screened for vectors containing inserts. The identity of the resulting plasmids was confirmed by sequencing. Plasmids for expression of His-tagged FBP were constructed as described above using plasmid DNA as the template and the forward primer 5'-GCAGGGAAAGTCATATGAAAACG-3' and the reverse primer 5'-CTCGAGCGCGTCCGGGTATTCGCGGATAAA-3' (engineered restriction sites underlined). These PCR products were ligated into pET20 (Novagen) digested with NdeI and XhoI and screened as described above. Plasmids are listed in Table 1.

Purification of FBP
Cultures (10 ml) of DM9297 containing an fbp-expression plasmid (Table 1), grown overnight in LB with 11 mM glucose, chloramphenicol, and ampicillin, were used to inoculate 1 liter of LB with 2.5 mM glucose, chloramphenicol, and ampicillin. The culture was grown at 37 °C with shaking (200 rpm) to an optical density at 650 nm of 0.6–0.7 and induced with 1 mM arabinose. After 3 h at 37°C, cells were harvested by centrifugation (7000 x g, 15 min, 4 °C). Cell pellets were resuspended in 15 ml of buffer A (50 mM sodium phosphate, 300 mM NaCl, 50 mM imidazole, pH 7.0) containing protease inhibitor mixture (Sigma P-8849) and disrupted with a French pressure cell (15,000 lb/inch2). The crude extract was clarified by centrifugation (39,000 x g, for 30 min at 4 °C). The clarified extract was loaded onto a HisTrap FF column (Amersham Biosciences) equilibrated with buffer A, then the column was washed with 15 column volumes of buffer A. Protein was eluted with a linear gradient (20 column volumes) from 50 to 500 mM imidazole in buffer A. Fractions containing FBP as judged by SDS-PAGE analysis were pooled, concentrated to 7–10 mg/ml, exchanged into buffer B (10 mM potassium phosphate, 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.4 mM fructose 1,6-bisphosphate, pH 7.0), frozen in liquid N2, and stored at –80 °C.

Assay of FBP Activity
FBP activity was determined using a coupled, spectrophotometric assay (37). Aliquots of FBP were thawed and diluted to 0.05 µg/ml in buffer C (10 mM potassium phosphate, 20% glycerol, pH 7.0) immediately prior to assay. Reactions (1 ml) contained: 100 mM Tris-Cl, pH 7.5, 0–100 µM Fru-1,6-P, 0–25 mM MgCl2, 2 mM ammonium sulfate, 0.2 mM NADP+, 50 µM EDTA, 5 mM beta-mercaptoethanol, 0.8 units of glucose-6-phosphate dehydrogenase, 0.8 units of phosphoglucose isomerase, and FBP. Reactions were initiated by the addition of FBP (250 ng), and the reduction of NADP+ was monitored at 340 nm ({epsilon} = 6.22 mM–1 cm–1). The linear steady-state portion of the A340 versus time plot was used to determine the kinetic parameters kcat, Km (Fru-1,6-P), and Hill coefficient by fitting initial velocity data to Equation 1 (R2 ≥ 0.99),

Formula 1(Eq. 1)
where {nu} is velocity, S is either the concentration of varied cofactor or substrate, Vmax is the velocity at saturating substrate and cofactor, Km is the Michaelis constant for the varied cofactor or substrate, and H is the Hill coefficient. The velocity data as plotted in the double-reciprocal form was fit by the following function,

Formula 2(Eq. 2)
where x is 1/[substrate], and y is 1/velocity. The initial slope is defined as b – ae, the slope of the asymptote is c/e, the y intercept is a, and the asymptote intercept is (be – c)/e2.

Inhibition by AMP was studied by using saturating Fru-1,6-P (100 µM), five concentrations of MgCl2 (0.5–8 mM), and five concentrations of AMP (0–8 µM for FBP(wt), 0–0.6 mM for FBP(T19I), and 0–8 mM for FBP(G20D) and FBP(T23I)). Inhibition by AICAR was studied by using saturating Fru-1,6-P (100 µM), five concentrations of MgCl2 (0.5–8 mM), and five concentrations of AICAR (0–120 µM for FBP(wt) and 0–10 mM for mutant FBP enzymes). Inhibition data were fit to the following equation (Equation 3) for nonlinear mixed-type inhibition (R2 ≥ 0.95),

Formula 3(Eq. 3)
where {nu} is the velocity, Vmax is the velocity with saturating substrate and cofactor with no inhibitor present, Ka is the Michaelis constant for Mg2+, S is the concentration of Mg2+, Kic is the competitive dissociation constant for inhibitor from the enzyme-inhibitor complex, Kiu is the uncompetitive dissociation constant for inhibitor from the enzyme-inhibitor complex, I is the concentration of inhibitor, and H is the Hill coefficient for Mg2+ derived from Equation 1. Linear and nonlinear regression analysis was preformed using SigmaPlot 9.0 (Systat Software).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Salmonella purH Mutants Are Unable to Use Glycerol as a Sole Carbon and Energy Source—The observation was made that a purH mutant strain of S. enterica (DM2) was unable to grow on minimal medium containing glycerol as sole carbon and energy source and supplemented with adenine and thiamine (Table 2). Further experiments demonstrated that this growth defect was eliminated by the addition of exogenous histidine. This finding led to the hypothesis that the accumulation of the purine biosynthetic intermediate, AICAR, was inhibiting an enzyme(s) required for growth on glycerol medium. AICAR is also generated as a byproduct of histidine biosynthesis; therefore addition of exogenous histidine reduces the intracellular concentration of AICAR in a purH mutant (5).


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  		TABLE 2
Mutations in fbp allow purH mutants to grow on minimal glycerol medium in the absence of histidine

 
Mutations That Allow a purH Mutant to Grow on Glycerol Medium in the Absence of Histidine Map to the fbp Locus—A purH mutant (DM2) was plated on minimal glycerol medium supplemented with adenine and thiamine and mutagenized with diethyl sulfate (3 µl spotted in the middle of a plate containing 108 colony forming units). Colonies appeared within 48 h and were concentrated in a ring around the spot of diethyl sulfate; 24 colonies were purified and re-tested for the glycerol-positive phenotype. One of these mutants was a purH+ revertant; and it was discarded. Twelve of the remaining strains were chosen at random for further study. The causative mutation in each was found to be genetically linked to a transposon located near the fbp locus. Six of these mutants were reconstructed and the fbp gene was amplified by PCR, cloned into pSU19, and sequenced. Three unique mutations were identified, causing the following amino acid substitutions: T19I, G20D, and T23I. Mutations causing the T19I and T23I substitutions were independently isolated multiple times. Isogenic strains containing each of the three mutations on the chromosome were constructed and tested for their ability to grow on minimal glycerol medium supplemented with adenine and thiamine (Table 2). The presence of any of these three mutations allowed a purH mutant to grow on minimal glycerol medium supplemented with adenine and thiamine. Strains containing null mutations in fbp also fail to grow on other gluconeogenic carbon sources such as acetate and succinate (19). In addition to glycerol, purH mutants failed to use acetate and succinate as sole carbon and energy sources in the absence of exogenous histidine (data not shown).

The Mutant FBP Enzymes Are Resistant to Inhibition by AICAR and AMP in Vitro—Wild-type and mutant FBP enzymes were purified as His6-tagged fusion proteins via Ni2+ affinity chromatography. The purified enzymes were judged to be ~90% pure by image analysis (Phoretix TotalLab) (Fig. 2). Kinetic parameters were determined from initial velocity data for the purified enzymes as described under "Experimental Procedures." Fig. 3 shows the {nu} versus substrate concentration for varying [Fru-1,6-P] (A.1) and [MgCl2](B.1) and the respective double reciprocal plots (A.2,B.2). These data as illustrated in the {nu} versus [Fru-1,6-P] and {nu} versus [Mg2+] plots are best fit by a modified Hill equation and the double reciprocal plots show data that are nonlinear and have a rising trend. This is characteristic of enzymes displaying positive cooperativity. To verify positive cooperativity, Hill plots were constructed for the assays with saturating Mg2+ and varied Fru-1,6-P (A.3) and the assays with saturating Fru-1,6-P and varied Mg2+ (B.3). Both plots are linear with a slope (n) corresponding to the Hill coefficient (H) derived from Equation 1. The kinetic parameters for the wild-type and variant S. enterica enzymes were derived using Equation 1 and are listed in Table 3. The values obtained for the wild-type enzyme were similar to those reported for the E. coli enzyme (20).


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  		TABLE 3
Kinetic parameters for wild-type and mutant FBP enzymes

Parameters are defined under "Experimental Procedures."

 
Inhibition by AMP and AICAR was examined for the wild-type and variant enzymes. The double-reciprocal plots of the {nu} versus [Mg2+] were found to be nonlinear and inhibition visually appeared to be competitive relative to [Mg2+]. The plots were nonlinear because of the cooperative behavior of Mg2+ binding to the enzymes. Linearity was obtained by raising the [Mg2+] to the power of the Hill coefficient of the specific enzymes, which was determined using Equation 1 (Table 3). A representative double reciprocal plot of wild-type FBP inhibition by AICAR is shown in Fig. 4. The type of inhibition exhibited for AMP and AICAR was determined by graphing initial rate data in the form of double reciprocal plots at various inhibitor concentrations and Dixon plots (1/{nu} versus [I]). By comparing actual data to theoretical values obtained by fitting the non-linear data to various inhibition equations it was found that the data are best described by Equation 3 for mixed-type inhibition. The inhibition constants are shown in Table 4. This means that AMP and AICAR bind to both the E and E·Mg complex. The high Kiu values and low Kic values obtained from the fits indicated that AMP and AICAR bind more tightly to the free enzyme and weaker to the E·Mg complex. Shown in the inset of Fig. 4 is a replot of the slopes for the theoretical data in Fig. 4 versus [AICAR]1. The relationship is linear indicating that AICAR and AMP (data not shown) do not inhibit the enzymes in a cooperative manner. As predicted from the phenotypic data, all three mutant forms of FBP had significantly higher Ki values than the wild-type enzyme (Kic ~ 120–1100-fold for AMP, ~45–60-fold for AICAR; Kiu ~ 100–1500-fold for AMP, 20–40-fold for AICAR).


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  		TABLE 4
Inhibition constants for wild-type and mutant Fpb enzymes

Parameters are defined under "Experimental Procedures."

 


Figure 2
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  		FIGURE 2.
Purification of wild-type FBP-His6 using a HisTrap FF column. Lane A, molecular weight marker; lane B, crude extract; lane C, clarified crude extract; lane D, flow-through; lane E, wash; lane F, elution.

 


Figure 3
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  		FIGURE 3.
Kinetic results demonstrate cooperative behavior for Salmonella FBP when Mg2+ and Fru-1,6-P are varied. The plots comparing the velocity of NADP+ reduction while varying the concentration of Fru-1,6-P (series A) or Mg2+ (series B) are shown. Plots are as follows: 1, {nu} versus [substrate or cofactor] fit by Equation 1; 2, double reciprocal plot of 1 fit by Equation 2; 3, Hill Plot fit using a linear regression (n = slope).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Results herein showed that the inability of S. enterica purH mutants to grow on gluconeogenic carbon sources in the absence of exogenous histidine is due to inhibition of FBP by the accumulation of the purine biosynthetic intermediate AICAR. Revertant mutations restoring growth of the purH mutant on glycerol mapped to fbp, and resulted in variants of FBP that were resistant to inhibition by AICAR and AMP.

The three variant proteins characterized in this work (T19I, G20D, and T23I) had lesions located in the AMP binding site of the FBP protein as determined from the crystal structure of the pig kidney enzyme (17). As shown in the partial alignment in Fig. 5, the AMP binding site is highly conserved between both bacterial and mammalian enzymes. S. enterica FBP is 97% identical and 98% similar to E. coli FBP, 43% identical and 63% similar to the pig kidney enzyme, and 44% identical and 63% similar to the human liver enzyme. Like the mammalian enzyme, but unlike the E. coli enzyme, FBP from S. enterica displayed cooperative kinetic behavior that had not been reported (mammalian hMg(II) ~ 2 (38)) (20). Interestingly, S. enterica FBP also displayed cooperative behavior for Fru-1,6-P that has not been reported for any FBP studied to date. Moreover, the S. enterica enzyme did not show cooperative kinetic behavior for AMP or AICAR inhibition, whereas this behavior has been shown for these inhibitors in the mammalian enzyme (mammalian hAMP = 2 (16, 38, 39)).


Figure 4
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  		FIGURE 4.
Inhibition of wild-type Salmonella FBP by AICAR. Double reciprocal plots of velocity versus [Mg2+]h at 0 mM (•), 15 mM ({circ}), 30 mM ({blacktriangledown}), 60 mM ({triangleup}), and 120 mM ({blacksquare}) AICAR. Lines represent plots of predicted 1/{nu} versus 1/[Mg2+] determined using nonlinear regression as described under "Experimental Procedures." Inset, replot of the slopes of the regression lines versus [AICAR].

 


Figure 5
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  		FIGURE 5.
Alignment of the N-terminal region of FBP homologs. Black shading indicates 100% conservation; gray shading indicates 50% conservation. Amino acid substitutions described in this work are indicated with letters above the alignment. Positions important for AMP inhibition in mammalian enzymes are indicated by pound signs. Asterisks mark every 20 amino acids, and the number 6 indicates conservation of a branched chain amino acid or methionine at that position. The figure was prepared with ClustalX (49) and GeneDoc (50).

 
Thr19 corresponds to Thr27 in the mammalian enzymes; the side chain of this residue contacts the phosphate group of AMP in the crystal structure (17), and replacement of Thr27 in the porcine enzyme with alanine increased the Ki for AMP 1300-fold (40). In this work, the T19I substitution increased the Kic for AMP 118-fold and the K 98-fold. Thr23iu corresponds to Thr31 in the mammalian enzymes; the side chain of this residue forms a hydrogen bond with the purine base of AMP in the crystal structure, and site-directed mutagenesis studies have confirmed its importance in AMP inhibition (39, 41). Gly20 corresponds to Gly28 of the mammalian enzymes, which forms a hydrogen bond to a water molecule in the AMP binding site but has not been studied by site-directed mutagenesis (42). Additional residues known to be important in AMP inhibition of the mammalian enzymes include Arg22, Ala24, Glu29, Ala54, Lys112, Tyr113, Gly122, and Arg140 (39–41, 43–45). A previous report on an AMP-insensitive fbp mutant of E. coli found that growth was unaffected by the presence of this mutation unless the allele was highly expressed, but further characterization of the enzyme was not reported (46).

The purine biosynthetic intermediate AICAR was shown to bind to the AMP binding pocket of human liver FBP by crystallographic studies (42) and shown to inhibit FBP in rat liver extracts (10, 11). All three substitutions described in this work significantly reduce inhibition of the enzyme for both AMP and AICAR. It is interesting to note that a perfect correlation does not exist between the effects of each substitution on inhibition by AMP and AICAR despite the fact that both compounds bind at the same site. In particular, the T23I substitution increases the Kic for AMP 1100-fold, but only increases the Kic for AICAR 31-fold. This observation may be explained by crystallographic data demonstrating that AICAR binds in a slightly different orientation than AMP; specifically, the imidazole ring of AICAR is rotated 180° relative to AMP in the crystal structure (42). The side chain of this residue forms hydrogen bonds to the purine ring and a water molecule in the AMP-bound structure, but in the AICAR-bound structure, the side chain hydrogen bond to the water molecule is replaced by a backbone nitrogen hydrogen bond from the same Thr residue. If the substitution of Ile for this Thr residue still allows the backbone hydrogen bond while eliminating the side chain hydrogen bond, this could explain the different effects of this substitution with respect to the two inhibitors.

The Kic determined for AICAR with the wild-type enzyme (37.3 ± 5.6 µM) agrees well with the IC50 determined by Iversen et al. (42) for the human liver enzyme. This is significant because it indicates that AICAR inhibits FBP at a physiologically relevant concentration. Bochner and Ames (47) determined the intracellular concentration of AICAR in Salmonella to be 105 µM in minimal glucose medium, whereas Rohlman and Matthews (2) measured concentrations of 40–150 µM in E. coli strain W3110. AICAR has many physiological effects in various organisms. These data suggest that in enteric bacteria AICAR has the potential to affect flux through gluconeogenesis, as FBP is a key control point for this pathway. The concentration of AICAR would be expected to decrease in response to exogenous purines or histidine due to feedback inhibition of the biosynthetic pathways for these compounds. It is known that AICAR levels increase significantly in response to folate stress (~3–6-fold) due to inhibition of the PurH enzyme (1, 2). Genetic evidence suggests that the PurH enzyme is sensitive to high temperature (48). Considering the in vitro kinetic parameters and in vivo pool sizes, it seems likely that flux through gluconeogenesis may be altered under growth conditions that impact the concentration of AICAR exemplified by blocking PurH by mutation.

In summary, the work described here has identified three amino acid substitutions in FBP from Salmonella that cause insensitivity to both AMP and AICAR. An additional potential regulatory role for the small molecule AICAR has been identified, although further work is necessary to determine whether this effect is physiologically relevant. Finally, the use of purH mutants could provide a powerful genetic selection for AMP-insensitive mutants of FBP, including substitutions that might not be predicted based on structural information.


   FOOTNOTES
 
* This work was supported in part by competitive Grant GM47296 from the National Institutes of Health and funds from a 21st Century Scientist Scholars Award from the J. S. McDonnell Foundation. 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. Back

1 Supported by Biotechnology Traineeship Grant T32 GM08349 from the National Institutes of Health, a Louis and Elsa Thomsen Wisconsin Distinguished Fellowship Award, and the William H. Peterson Predoctoral Fellowship from the Department of Bacteriology. Present address: Div. of Chemistry and Chemical Engineering, 210-41, California Institute of Technology, Pasadena, CA 91125. Back

2 To whom correspondence should be addressed: 420 Henry Mall, Madison, WI 53706. Tel.: 608-265-4630; Fax: 608-262-9865; E-mail: downs{at}bact.wisc.edu.

3 The abbreviations used are: AICAR, 5-amino-4-imidazole carboxamide ribotide; FBP, fructose-1,6-bisphosphatase; Fru-1,6-P, fructose 1,6-bisphosphate; Fru-2,6-P, fructose 2,6-bisphosphate. Back


   ACKNOWLEDGMENTS
 
We acknowledge P. Frey and W. W. Cleland (Department of Biochemistry, University of Wisconsin-Madison) for advice on the kinetic analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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