Purification and Characterization ofglpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli

E. coli strains and cloning of glpX. E. coli strains used in this study are listed in Table1. Strains were grown in Luria broth (LB) supplemented with antibiotics as needed or in minimal medium (7) containing 0.4% glycerol or 0.2% glucose.

The glpX gene was PCR amplified from chromosomal DNA of strain MG1655 using the primer pair acgtgaaTTCCCCTGTGCTACACTTCG (hybridizes to a region 48 bp upstream of the initiation codon) and acgttctagaTTGCCTGTTACCCAATCAGC (hybridizes to a region 102 bp downstream of the termination codon), containing sequence mismatches (lowercase) and restriction sites EcoRI andXbaI (underlined). The PCR product was ligated intoXbaI/EcoRI-cut pZE14 (18) forming pJB100. This plasmid was introduced into strain DF657 (Δfbp) to test for complementation of the Fbp⁻ (glycerol-negative) phenotype. DF657(pJB100) was capable of slow growth on glycerol. Therefore, increased expression ofglpX from the high-copy-number plasmid pJB100 complements the Fbp⁻ phenotype.

Overexpression and purification of FBPase II.To facilitate overexpression and purification of FBPase II, a pT7-7 (28) derivative containing glpX was constructed (pJB300B). TheNdeI-SalI fragment that was inserted into the same sites of pT7-7 to form pJB300B was obtained by PCR amplification using primers acgtaccaattgaggagatatacaTATGAGACGAGAACTTGCCATC and acgtgtcgacTGCCTTATCTTCGTTCTCCG, with DNA from strain MG1655 as the template (sequence mismatches are lowercase and restriction sites are underlined). The expected nucleotide sequence of glpX in pJB300B was confirmed. Overexpression of glpX was induced in JB108(pJB300B) with 200 μM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h in a 200-ml LB culture with an optical density at 600 nm of 0.6. Cells were harvested by centrifugation, resuspended in 2.5 ml of 20 mM Tricine (pH 7.7)–50 mM KCl–1 mM MgCl₂–1 mM dithiothreitol (DTT)–0.5 mM EDTA, lysed by sonication, and centrifuged at 140,000 × g for 30 min. Nucleic acids were precipitated by addition of polyethylenimine (0.05%, vol/vol) and centrifugation at 10,000 × g for 20 min. FBPase II activity was precipitated by addition of 0.4 g of (NH₄)₂SO₄ ml⁻¹. The (NH₄)₂SO₄ pellet was dissolved in 2.3 ml of 50 mM Tris-HCl (pH 7.7)–0.2 mM MgCl₂–0.1 mM EDTA and fractionated by anion exchange chromatography on a Q HR15 column (Waters) at room temperature using a gradient of 0.075 to 0.5 M NaCl in 50 mM Tris-HCl (pH 7.7)–0.2 mM MgCl₂–0.1 mM EDTA. Peak fractions were pooled and precipitated with 0.5 g of (NH₄)₂SO₄ ml⁻¹, redissolved, and then dialyzed against 20 mM Tricine (pH 7.7)–1 mM MgCl₂–0.1 mM DTT–15% glycerol. All steps of the purification procedure were monitored by coupled spectrophotometric assay. A total of 2.8 mg of protein was purified 4.4-fold, with a 55% recovery and specific activity of 4.2 U mg⁻¹. Analysis of purified product by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was >95% pure. Protein concentrations were determined as described by Bradford (5) with bovine serum albumin as the standard.

Optimal conditions for enzyme activity.Using a coupled spectrophotometric assay (Fig. 1), the highest activity was obtained with 8 μg of FBPase II in a 1-ml reaction mixture containing 0.5 to 2 mM MnCl₂, 50 to 125 mM KCl, 0.02 M Tricine (pH 7.7), and 1.5 mM Fru(1,6)P₂. Omission of Mn²⁺resulted in >90% loss of activity. Replacing Mn²⁺ with 1 mM ZnCl₂, CaCl₂, FeSO₄, or CuSO₄ resulted in almost complete loss of activity. Replacement with 3 to 10 mM MgCl₂ resulted in <10% of the activity present with Mn²⁺. The presence of 1 mM DTT had no effect on FBPase II activity.

• Open in new tab
• Download powerpoint

Fig. 1.

(A) Dependence of FBPase II activity on concentration of Fru(1,6)P₂. Each data point is presented as an average of two to four determinations, with standard errors (error bars). Coupled spectrophotometric assay was used with a solution containing 8 μg of FBPase II, 0.2 U of phosphoglucose isomerase, 0.4 units of glucose-6-phosphate dehydrogenase, 20 mM Tricine (pH 7.7), 50 mM KCl, 1 mM MnCl₂, and 0.25 mM NADP in a final volume of 1 ml at room temperature (24). The rate of reduction of NADP to NADPH was determined by monitoring absorbance at 340 nm. (B) Lineweaver-Burk replot of selected data points from panel A.

Molecular mass determination.The molecular mass of the GlpX subunit estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 40 kDa, which is near the expected size based on the deduced amino acid sequence (36 kDa). Gel filtration chromatography was performed using a Waters Protein Pak Glass 300SW column in 20 mM Tricine (pH 7.7)–100 mM KCl with 1 mM MnCl₂ or 1 mM MgCl₂. Standards included ribonuclease A (17 kDa), carbonic anhydrase (29 kDa), ovalbumin (45 and 90 kDa), bovine serum albumin (66 and 132 kDa) and phosphorylase b (97.4 kDa). Times of protein elution were determined by monitoring the absorbance at 280 nm. At high, medium, and low concentrations of FBPase II (320, 160, and 40 μg of FBPase II, respectively, in 200 μl of loading volume), the protein eluted at 90, 80, and 47 kDa, respectively. FBPase II is most likely a dimer at higher concentrations, in contrast to the tetramericfbp-encoded FBPase I (1).

Catalytic properties.A K_m of ∼35 μM and V_max of ∼3.3 U mg⁻¹ were determined for FBPase II using Fru(1,6)P₂ levels below 0.25 mM (Fig. 1). Substrate inhibition was observed at high Fru(1,6)P₂ concentrations. FBPase II has lower affinity for Fru(1,6)P₂ compared to FBPase I (apparent K_m of 5 μM [1]). Based on specific activities of the purified enzymes, the deduced turnover number for FBPase II is about seven times less than that of FBPase I (19).

Substrate specificity of FBPase II.Substrate specificity was determined by measuring the rate of enzyme-catalyzed production of inorganic phosphate from various potential substrates (see footnoted to Table 2). Fru(1,6)P₂ was the best substrate found, while fructose 1-phosphate and ribulose 1,5-bisphosphate served less well as substrates. The apparentK_m and V_max of FBPase II with fructose 1-phosphate were 1 mM and 1.4 U mg⁻¹, respectively, giving aV_max/K_m almost 70 times lower than that obtained using Fru(1,6)P₂. Ribulose 1,5-bisphosphate at 1 mM produced 15% of the phosphatase activity obtained with 1.5 mM Fru(1,6)P₂. Glucose 6-phosphate, fructose 6-phosphate, mannose 6-phosphate, glucose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate, sorbitol 6-phosphate, and glycerol-P were not significant substrates (all present at 1 mM [data not shown]).

Effectors of FBPase II.Fructose 1-phosphate, inorganic phosphate, ADP, and ATP inhibited FBPase II activity (Table2). Fructose 1-phosphate and inorganic phosphate inhibited activity competitively, with apparentK_is of 1 and 0.35 mM, respectively. Fructose, fructose 6-phosphate, and glucose 6-phosphate were not inhibitors (data not shown). AMP had no effect on enzyme activity, but ATP and especially ADP inhibited activity at low Fru(1,6)P₂ (≤0.1 mM). PEP almost doubled enzyme activity (Table 2), while addition of dihydroxyacetone phosphate, glycerol, or glycerol-P (all at 1 mM) did not affect activity (not shown). The response of FBPase II to effector molecules PEP, ADP, and ATP associates it with many enzymes of the Embden-Meyerhof pathway that are regulated by PEP, Fru(1,6)P₂, ATP, ADP, or AMP (9). The properties of FBPase II are distinct from those of FBPase I, an enzyme exquisitely sensitive to inhibition by AMP (50% inhibition by 15 μM AMP [1]). AMP inhibition of FBPase I is alleviated by PEP, although higher concentrations of PEP are inhibitory (>1 mM PEP).

Physiological role of glpX.Genetic studies were undertaken in an attempt to discern the physiological role ofglpX. First, the roles of the two FBPases were investigated using congenic strains with the four possible combinations of wild-type and defective fbp and glpX alleles: JLD2402 (Δfbp glpX::Spc^r), JLD2403 (fbp⁺glpX::Spc^r), JLD2404 (Δfbp glpX⁺), and JLD2405 (fbp⁺glpX⁺) (Table 1). Thefbp⁺glpX::Spc^rstrain grew as well as the wild type on LB or glucose, fructose, succinate, or glycerol minimal medium, aerobically or anaerobically. Apparently, FBPase II is not crucial to cell growth under these conditions when FBPase I is present. Strains lacking FBPase I (e.g.,Δfbp glpX⁺ strains DF657 and JLD2404) grew normally on glucose or fructose medium but were unable to grow on minimal medium supplemented with glycerol or other gluconeogenic substrates, indicating that chromosomal glpX⁺does not compensate for the loss of fbp expression. (However, increased expression of glpX from multicopy plasmid pJB100 complemented the Fbp⁻ phenotype.)

The Δfbp glpX⁺ strain (JLD2404) was able to revert to slow growth on glycerol minimal medium enriched with 0.03% Casamino acids and 2.5 mM KNO₃ (with or without O₂). These revertants lost the ability to grow on glycerol when transduced to glpX::Spc^r. The Δfbp glpX::Spc^r strain JLD2402 did not show any reversion under the same conditions. A possible explanation for reversion of Δfbp glpX⁺strains to a glycerol-positive phenotype is a mutation yielding elevated glpX expression.

The effect of the glpX and fbp mutations on glycogen accumulation was tested by exposure of the four congenic strains to iodine vapor following growth on Kornberg medium containing 0.4% glycerol instead of glucose (17). Glycogen accumulated to higher levels in both fbp⁺ strains (maroon colony color) compared to levels found in the Δfbp mutants (orange colony color). Therefore, accumulation of glycogen to wild-type levels requires FBPase I but is not affected by the glpXmutation.

We hypothesized that the inability of Δfbp glpX⁺ strains to grow on gluconeogenic substrates may be due to low-level expression of glpX combined with competing hexose catabolic pathways that would prevent accumulation of hexose sufficient for cell growth. To test this possibility, theΔfbp allele was introduced by cotransduction withzjg920::Tn10 into a pfkA2strain deficient in glycolysis (phosphofructokinase I; strain AM1) and into an edd gnd strain deficient in the Entner-Douderoff pathway (6-phosphogluconate dehydrase and 6-phosphogluconate dehydrogenase; strain R6). In both cases, transductants unable to grow on glycerol were obtained at the expected frequency, demonstrating thatglpX function is insufficient to provide hexose for growth of these strains where presumed competing pathways are impaired. We did observe that a pfkA glpX::Spc^r strain grew more slowly with glucose or glycerol than did the pfkA glpX⁺ parent strain AM1, indicating a functional importance of FBPase II in this strain.

Three classes of FBPases.Bacterial FBPases are members of three different orthologous groups (30), including a group of glpX-encoded FBPases (class II). Some bacteria that contain orthologs of FBPase II include Klebsiella aerogenes(91% identity), Yersinia pestis (81%), Haemophilus influenzae (67%), Bacillus subtilis (49%),Clostridium acetobutylicum (45%), Mycobacterium tuberculosis (43%) and Synechococcus sp. strain PCC7942 (39% [29]). There is little similarity of these class II FBPases to the other two orthologous groups of FBPases, those of FBPase I (E. coli) (class I), and those of a very divergent FBPase of B. subtilis (class III [11]). From the sequence data available, no bacterial genome has a combination of class I and class III FBPases, although combinations of classes I and II and of classes II and III are seen.

In conclusion, glpX-encoded FBPase II is still somewhat of an unknown entity in the overall framework of E. colimetabolism. FBPase II is conserved in many other bacteria and is the only known FBPase in some organisms (e.g., M. tuberculosis). The enzyme is distinct in many aspects from FBPase I. However, the specificity and high affinity of FBPase II for substrate and its regulation by PEP and ADP suggest that the enzyme functions with FBPase I in the central pathways of carbohydrate metabolism.