Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Bacterial strains, plasmids, primers, and growth media.The bacterial strains, primers, and plasmids used in this study are described in Table 1. E. coli TCM15 is a derivative of BW30270, E. coli K-12 MG1655 rph⁺ fnr⁺, in which the gutQ and kdsD genes were disrupted via the phage λ Red recombinase system (6, 12). All strains were grown in lysogeny broth (LB) medium (13). TCM15 cultures were supplemented with A5P (15 μM) and d-glucose-6-phosphate (G6P; 10 μM). A5P is necessary to complement the API defect, and G6P induces the transporter necessary for the uptake of A5P (8).

Cloning, expression, and purification of B. fragilis Q5LIW1.The YP_209877.1 gene, referred to here by the GenBank ID YP_209877.1, encoding Q5LIW1, was amplified from the genomic DNA of Bacteroides fragilis NCTC 9343 using the primers BF0137START and BF0137STOP (Table 1), which were designed to incorporate NdeI and BamHI sites. PCR products were purified via extraction from a 1% (wt/vol) agarose gel using a Qiagen QIAquick gel extraction kit, inserted into vector pCR2.1-TOPO using the TA TOPO cloning kit, and subcloned, after digestion of the insert-containing vector with NdeI and BamHI, into similarly restricted expression vector pT7-7 (14). The resulting plasmid, pT7-7-YP_209877.1, was transformed into E. coli TOP10 chemically competent cells. DNA sequencing of the resulting plasmid, pT7-7-YP_209877.1, confirmed that the YP_209877.1 gene sequence was identical to the sequence published in the NCBI entry. This plasmid was subsequently transformed into E. coli Rosetta 2 (DE3) pLysS chemically competent cells. A fresh transformant was grown in LB medium supplemented with 100 mg/liter ampicillin and 30 mg/liter chloramphenicol at 37°C while being shaken at 250 rpm until the optical density at 600 nm reached 0.6. The culture was cooled to 19°C, and expression of protein was induced with the addition of isopropyl-β-d-1-thiogalactopyranoside to 0.42 mM. After 16 h of incubation at 19°C, cells were harvested by centrifugation (6,000 × g, 10 min, 4°C). The pellet was suspended in 25 ml buffer B (40 mM Tris-HCl, pH 7.5), sonicated on ice (3 cycles of 20-s bursts, 2-min pauses between pulses), and clarified by centrifugation at 18,000 × g for 30 min. Solid ammonium sulfate was slowly added, while the mixture was being stirred, to the clarified lysate to reach 50% saturation at 4°C, and stirring was continued for an additional 10 min. Precipitated proteins were removed by centrifugation at 15,000 × g for 25 min, and solid ammonium sulfate was slowly added to the supernatant to reach 60% saturation (at 4°C) while the mixture was being stirred. The solution was stirred for an additional 10 min after the final addition of ammonium sulfate. Q5LIW1 was pelleted, along with some contaminating proteins, by centrifugation at 15,000 × g for 25 min and resuspended in buffer A (40 mM Tris-HCl, pH 7.5, 1.4 M ammonium sulfate) prior to being passed through an 0.22-μm Millipore polyvinylidene difluoride (PVDF) filter. The solubilized protein was loaded onto a phenyl-Superose column, which had been equilibrated with buffer A. Protein was eluted with an inverse linear gradient of 40 to 100% (vol/vol) buffer B in buffer A over 15 column volumes at 0.5 ml/min. Fractions containing Q5LIW1, as determined by SDS-PAGE, were pooled and concentrated using an Amicon ultracentrifugal filter (10,000-molecular-weight cutoff [MWCO]), and buffer was exchanged into 40 mM Tris-HCl, pH 7.5, and 25% (vol/vol) glycerol and stored at −80°C.

Site-directed mutagenesis, expression, and purification of E. coli kdsDΔ2CBS.Plasmid template DNA (pT7-7-yrbH, approximately 0.5 pmol) was added to a PCR cocktail containing 1× PfuTurbo buffer, 200 μM (each) deoxynucleoside triphosphate (dNTP), 3 μM EcKdsDΔ2CBS.F and EcKdsDΔ2CBS.R, and 2.5 U PfuTurbo DNA polymerase. The reaction mixture was incubated in an MJ Research PTC-200 thermal cycler with standard site-directed mutagenesis parameters. The reaction mixture was digested with DpnI for 1 h at 37°C and purified via extraction from an agarose gel. The resulting plasmid, pT7-7-EcKdsDΔ2CBS, was transformed into E. coli TOP10 chemically competent cells. DNA sequencing of the plasmid pT7-7-EcKdsDΔ2CBS confirmed that the kdsD gene had been truncated to include a stop codon between the SIS domain and first CBS domain (resulting in a gene encoding a 213-amino-acid protein). The plasmid was subsequently transformed into E. coli BL21(DE3) chemically competent cells. A fresh transformant was grown, and cells were harvested as described above. EcKdsDΔ2CBS was purified via a Hi-Load (16/10) Q-Sepharose fast-flow column, followed by ammonium sulfate precipitation as previously described (5).

Molecular mass determination.The subunit mass of Q5LIW1 was determined via electrospray ionization mass spectrometry utilizing an LCT electrospray/time of flight spectrometer. The native molecular mass of Q5LIW1 was estimated by gel filtration chromatography on a HiPrep (26/60) Sephacryl S-100 column. Standards, run in triplicate, included bovine serum albumin (BSA) dimer (132.4 kDa), BSA monomer (66.2 kDa), chicken egg white ovalbumin monomer (44.3 kDa), and cytochrome c (12.4 kDa). A log(molecular mass)-versus-elution volume/void volume (V_e/V_o) plot was fitted by linear regression, using Microsoft Excel. The molecular mass of Q5LIW1 was determined experimentally by measuring its elution volume and calculating the log(molecular mass) from the standard curve.

Metal content analysis.Enzyme samples were prepared for metal content analysis by 24 h of dialysis at 4°C against 2 liters of metal-free 40 mM Tris-HCl (pH 8.5) buffer and stored in metal-free glass vials. The divalent metal content, of each sample and of the buffer used in dialysis as a control, was determined using high-resolution inductively coupled plasma mass spectrometry on a Finnigan MAT Element instrument at the University of Michigan Department of Geology.

Substrate specificity.To determine the ability of recombinant Q5LIW1 to convert aldoses to ketoses, the enzyme was assayed with various aldoses, including A5P, G6P, d-glucose-1-phosphate, d-ribose-5-phosphate, d-arabinose, d-mannose-6-phosphate, d-glucosamine-6-phosphate, and d-ribose using the discontinuous cysteine-carbazole assay (5). For each potential substrate, in triplicate, a 50-μl solution of 100 nM enzyme, 100 mM 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris propane), pH 8.5, and 10 mM substrate was incubated for 10 min at 37°C before being quenched with an equal volume of 25 N H₂SO₄. A 90-μl aliquot was transferred to a separate microplate containing a freshly prepared cysteine-carbazole assay mixture (10 μl of a 0.12% ethanolic carbazole solution, 10 μl of 1.5% aqueous cysteine-HCl, and 230 μl of 25 N H₂SO₄) and incubated at room temperature (∼21°C) for 3 h to develop color. Enzyme substrates were identified by comparing the color of the reaction to the color of a no-enzyme control.

Complementation of a kdsD/gutQ defect in E. coli.Electrocompetent cells were prepared, from the E. coli TCM15 variant that carries a kanamycin resistance cassette, by growing the cells in LB medium until early log phase (optical density at 600 nm, ∼0.5). The cells were then harvested via centrifugation and washed three times in ice-cold 10% glycerol (8). Empty pT7-7 vector, pT7-yrbH, pT7-7-EcKdsDΔ2CBS, pT7-7-c3406, and pT7-7-YP_209877.1 were separately transformed into electrocompetent E. coli TCM15 cells. Transformed cells were grown on LB agar plates supplemented with 100 mg/liter ampicillin, 50 mg/liter kanamycin, 15 μM A5P, and 10 μM G6P. Liquid LB medium supplemented with the same concentrations of antibiotics, A5P, and G6P was inoculated with single colonies, which were grown overnight at 37°C. Cells were washed twice, by centrifugation at 3,300 × g and resuspension in liquid LB medium, to remove A5P and G6P in the overnight culture; streaked on LB/agar plates with and without A5P/G6P; and incubated overnight at 37°C. Genes that complemented the API defect allowed the transformed cells to grow both with and without added A5P/G6P.

Determining the pH rate profile of Q5LIW1.The optimal pH for Q5LIW1 was determined by assaying the enzymatic activity of Q5LIW1, using the discontinuous cysteine-carbazole assay, in a series of buffer solutions of various pHs. Buffer solutions (200 mM buffer, 2 mM EDTA) were prepared in 0.25 pH increments from 5.0 to 9.5 at 37°C. 2-(N-Morpholino)ethanesulfonic acid (MES) buffer was used from pH 5.0 to 6.0, and Bis-Tris propane buffer was used from pH 6.25 to 9.5. Final reaction concentrations were 100 mM buffer, 1 mM EDTA, 10 mM A5P, and 400 nM enzyme. Reaction mixtures were incubated at 37°C for 3 min before being quenched with equal volumes of 25 N H₂SO₄. Quenched reactions were developed, as described above, and incubated at room temperature (∼21°C) for 3 h before the absorbance was measured at 540 nm. The enzymatic activity at each pH was determined in triplicate, and reaction rates were determined by fitting time points using linear regression.

Enzyme kinetics.Kinetic parameters for the isomerization of A5P to Ru5P or G6P to d-fructose-6-phosphate (F6P) catalyzed by Q5LIW1 and EcKdsDΔ2CBS were determined, in triplicate, using the discontinuous cysteine-carbazole assay with 100 nM Q5LIW1 or EcKdsDΔ2CBS, 100 mM Bis-Tris propane (pH 8.5), 1 mM EDTA, and substrate concentrations ranging from 0.156 mM to 20 mM A5P or 0.195 mM to 25 mM G6P. Individual assay mixtures, which were preheated at 37°C for 3 min before the addition of enzyme to initiate the reaction, and appropriate controls were allowed to react for 3 min at 37°C before being quenched with equal volumes of 25 N H₂SO₄. The quenched reaction mixtures were developed as described above. Control reactions, with Ru5P/F6P, showed that less than 10% of the substrate was converted to product under all conditions tested. Kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation using nonlinear least-squares regression with GraphPad Prism 5 software.

Assay of A5P isomerase activity.Kinetic parameters for the isomerization of Ru5P to A5P were determined, in triplicate, using a modified coupled Aminoff assay utilizing the 3-deoxy-d-manno-octulosonate 8-phosphate synthase (Kdo8PS) from Arabidopsis thaliana (15, 16). Reaction mixtures containing final concentrations of 10 mM phosphoenolpyruvate (PEP), 0.15 mg/ml Kdo8PS, 100 mM Bis-Tris propane (pH 8.5), and 1 mM EDTA and final concentrations of Ru5P ranging from 0 to 10 mM were heated separately from mixtures of Q5LIW1 or EcKdsDΔ2CBS (100 nM final concentration) to 37°C for 3 min. The reaction was then initiated by the addition of Q5LIW1 or EcKdsDΔ2CBS, and the reaction mixture was incubated for 2 min at 37°C. Reactions were quenched with an equal volume of 10% (wt/vol) trichloroacetic acid. To develop the color, 50 μl of each reaction mixture was transferred to a separate glass tube, after which 100 μl of a solution containing NaIO₄ (25 mM) and H₂SO₄ (0.125 N) was added, followed by incubation for 10 min at room temperature (∼21°C). The addition of 200 μl of a solution containing NaAsO₂ (2%, wt/vol) and HCl (0.5 N) neutralized the excess NaIO₄. After the disappearance of the color, a solution containing 500 μl of 0.36% thiobarbituric acid, pH 9.0, was added and the reaction mixture was incubated at 95°C for 10 min. These mixtures were transferred to a 96-well flat-bottom plate, and the absorbances were read at 549 nm. Data were fitted using the nonlinear least-squares regression function of GraphPad Prism 5 software.

d-Glucose-6-phosphate isomerase assays.To determine the kinetic parameters for the isomerization of F6P to G6P catalyzed by Q5LIW1, a coupled assay was used, in which the G6P produced by Q5LIW1 was oxidized to 6-phosphoglucono-δ-lactone by glucose-6-phosphate dehydrogenase and the cofactor NADP⁺. The formation of NADPH was measured at 340 nm, which allowed for reaction progress to be monitored (17). Individual reactions were performed in a quartz cuvette, in triplicate. The coupling enzyme (E. coli d-glucose-6-phosphate dehydrogenase, gene name zwf) was diluted into 2× reaction buffer (200 mM Bis-Tris propane, pH 8.5, 2 mM EDTA) to a final coupling enzyme concentration of 1 μM. NADP⁺ (0.16 mM final concentration) and F6P (0.01 to 3 mM final concentration) were subsequently added to the reaction mixture, which was allowed to incubate for 1 min at room temperature (∼21°C). The reaction was initiated by the addition of Q5LIW1 (final concentration, 100 nM), and the absorbance, at 340 nm, was monitored at 10-s intervals for 5 min, using a Hewlett-Packard 8453 diode array spectrophotometer. Initial reaction rates (nM/s) were determined by analysis of progress curves via linear regression. Kinetic parameters were obtained by fitting of the rate versus substrate concentration data, from assays performed in triplicate, to the Michaelis-Menten equation using nonlinear least-squares regression in GraphPad Prism 5.

Generation of CMP-Kdo.CMP-Kdo was generated using E. coli CMP-Kdo synthetase (KdsB) (18). In a typical reaction, CTP (0.55 mM) and Kdo (1.1 mM), were added to 4× reaction buffer (400 mM Bis-Tris propane, pH 8.5, 5 mM MgCl₂), and the reaction was initiated by addition of KdsB to a final concentration of 13.2 μg/ml. The reaction was allowed to incubate for 10 min at room temperature before being quenched with EDTA (45 mM final concentration).

The Eikonogen assay (19, 20), which allows for the direct correlation of the concentration of CMP-Kdo with the amount of inorganic pyrophosphate produced, was used to determine the concentration of inorganic pyrophosphate produced. In this assay, the CMP-Kdo solution (50 μl) was treated with 2.5% ammonium molybdate (50 μl), 0.5 M β-mercaptoethanol (50 μl,) and Eikonogen reagent (20 μl, 0.125 g sodium sulfite, 7.325 g sodium metabisulfite, and 0.125 g 1-amino-2-naphthol-4-sulfonic acid in 50 ml hot deionized water). The mixture was incubated at room temperature (∼21°C) for 30 min, and the absorbance was read at 540 nm. A malachite green assay, as previously described, was used to correct for inorganic phosphate concentrations (20). Absorbances were compared to a standard curve prepared using a serial dilution of a standard aqueous solution of Na₂P₂O₇. Data were analyzed by linear regression in Microsoft Excel.

Inhibition kinetics with CMP-Kdo.To test if recombinant Q5LIW1 was inhibited by CMP-Kdo, a coupled assay in which the Ru5P produced by Q5LIW1-catalyzed isomerization of A5P was reduced to ribitol using an excess of NADPH and the enzyme Bcs1 (CDP-ribitol synthase) was performed. Reaction mixtures containing A5P (0.25 mM or 0.50 mM), Bis-Tris propane buffer (100 mM; pH 8.5), EDTA (1 mM), NADPH (0.16 mM), and Bcs1 (96 μg/ml) (21) were incubated separately from mixtures containing Q5LIW1 (100 nM) and CMP-Kdo (final assay concentrations ranging from 0 to 68.51 μM) for 3 min at 37°C. The enzyme-inhibitor mixture was added to the reaction mixture, mixed for 20 s, and incubated at 37°C for 10 min while the absorbance at 340 nm was monitored every 24 s using a SpectraMax M5 microplate reader (Molecular Devices). Absorbance values were plotted versus time in Microsoft Excel and fitted using linear regression. The slopes were converted to reaction rates in nM/s. Rates were plotted as a function of inhibitor concentration in GraphPad Prism 5 and fitted using the nonlinear least-squares technique to a model of competitive inhibition based upon the Michaelis-Menten equation. Rates were converted to percent response of maximum, and the log(response)/(1 − response) was plotted versus log[CMP-Kdo] in Microsoft Excel to generate a Hill plot (see Fig. 6B).

Equilibrium constant (K_eq) determination.Solutions containing 100 mM Bis-Tris propane buffer, pH 8.5, 1 mM EDTA, 10% D₂O, 500 nM Q5LIW1 or EcKdsDΔ2CBS, and a 5 mM concentration of F6P, G6P, A5P, or Ru5P were incubated at room temperature (∼21°C) for 72 h, sufficient to reach equilibrium. These solutions were analyzed by ³¹P nuclear magnetic resonance (NMR) using a Varian 400 multinuclear NMR spectrometer. A solution of 0.05 N phosphoric acid standard was sealed within a capillary tube and set to a value of 0 ppm. Spectra were acquired using 64 scans with a 10-s relaxation delay between scans. Preliminary studies with longer relaxation times did not show a change in peak integrations, which supports the assumption that the chosen delay time was greater than three times the T1 relaxation parameter for G6P, F6P, A5P, and Ru5P.

Effect of divalent metal ions on the API activity of Q5LIW1.To determine the effect of divalent metal ions on the API activity of Q5LIW1, samples of Q5LIW1 were diluted in buffer containing 108.2 mM Bis-Tris propane, pH 8.5, and 10.82 μM EDTA or divalent metal salt and incubated on ice for 30 min. The API activity was assayed using the Aminoff assay (15). Final concentrations in each reaction mixture were 100 nM Q5LIW1, 100 mM Bis-Tris propane (pH 8.5), 0.15 mg/ml KdsA, 10 mM PEP, and 5 mM Ru5P. Reaction mixtures were incubated for 3 min at 37°C before being quenched with an equal volume of 10% (wt/vol) trichloroacetic acid, and the color was developed as described above in the A5P isomerization assay. Metal salts tested included BaCl₂ · 2H₂O, MnCl₂ · 4H₂O, ZnCl₂, NiCl₂ · 6H₂O, CoSO₄ · 7H₂O, CuSO₄, FeSO₄ · 7H₂O, CdCl₂, MgCl₂, CaCl₂ · 2H₂O, and HgCl₂.