Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB) In Vivo Justified by Intracellular Amino Sugar Metabolite Concentrations

Abstract

We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme in E. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P, and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drives E. coli NagB (NagBEc) into its high activity state, as observed during growth on GlcN (L. I. Álvarez-Añorve, I. Bustos-Jaimes, M. L. Calcagno, and J. Plumbridge, J Bacteriol 191:6401-6407, 2009, http://dx.doi.org/10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of the nag genes, from its binding sites, so there is only a small increase in nagB expression. We replaced the gene for the allosteric NagBEc enzyme with that of the nonallosteric, B. subtilis homologue, NagBBs We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor did we detect any effect on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB.

Importance: Chitin, the polymer of N-acetylglucosamine, is an abundant biomaterial, and both glucosamine and N-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules, including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control, and N-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization in Escherichia coli Here, we investigate how the metabolic status of the bacteria impacts on the activity of NagBEc and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.

FIG 1

Use of amino sugars by E. coli. (A) Organization of the divergent nagE and nagBACD operons encoding the PTS transporter of GlcNAc (NagE), the enzymes for the metabolism of GlcNAc, GlcNAc6P deacetylase (NagA), and GlcN6P deaminase (NagB), the repressor of the operon (NagC) and NagD, a UMP phosphatase (1). (B) Metabolism of amino sugars. GlcNAc predominantly enters the bacteria by the phosphotransferase system (PTS) transporter, NagE, producing intracellular GlcNAc6P, which is metabolized by the NagA (GlcNAc6P deacetylase) and NagB (GlcN6P deaminase) enzymes to fructose-6P (F6P). GlcN is transported by the manXYZ-encoded PTS transporter, producing GlcN6P, which is also metabolized by NagB. Glucose is predominately transported by the ptsG-encoded PTS transporter giving G6P and is converted to F6P, the first common compound of the glycolytic pathway. GlcN6P is synthesized by the glmS-encoded enzyme and converted, by the sequential action of the glmM and glmU-encoded enzymes, to UDP-GlcNAc, the essential precursor to the amino sugar components of the peptidoglycan (PG) and lipopolysaccharide.

FIG 2

Effect of growth on amino sugars on glycolysis. A schematic view of glycolysis and the TCA cycle during growth on Glc, GlcN, or GlcNAc is shown. The intracellular concentrations of the compounds indicated are shown for the three strains: MC-B1 expressing wild-type NagB_Ec (strain 1), MC-B362 expressing GamA (strain 2), and MC-B364 expressing NagB_Bs (strain 3). The concentrations of metabolites during growth on glucose (Glc) are shown in blue, those on GlcN are shown in green, and those on GlcNAc are shown in brown/red. Glyceraldehyde-3P (GAP) and pyruvate (PYR) are unstable during the extraction process and were not quantified. The complete analysis was repeated twice. A representative experiment is shown with means and standard deviations of three replicate filters. Data for GlcN6P and GlcNAc6P are expanded in Fig. 3.

FIG 3

Effect of growth on glucose (Glc), GlcN, and GlcNAc on the pools of GlcN6P and GlcNAcP in the wild type, in nagB recombinants expressing the B. subtilis GlcN6P deaminases, and in strains with the nagA and nagB genes deleted. Intracellular concentrations of GlcN6P and GlcNAc6P during growth of wild-type E. coli NagB_Ec (MC-B1), E. coli expressing GamA (MC-B362), and E. coli expressing NagB_Bs (MC-B364) in Glc (A), GlcN (B), and GlcNAc (C) and the ΔnagA (MC-B174c) and ΔnagB (MC-B76c) deletion strains in glucose are shown. The results are means with the standard deviations for two biological replicate cultures (six filters). Note the change in scales for concentrations. The same data are given in numerical form in Table S2 in the supplemental material.

FIG 4

Activation of NagB_Ec by subsaturating amounts of its allosteric activator, GlcNAc6P. (A) Enzyme activity expressed as v/V (fraction of V_max measured as initial velocity divided by maximum velocity) was plotted versus the GlcN6P concentration at the GlcNAc6P concentrations shown. The v/V ratio is equivalent to the fraction of substrate saturation, ȳ_S, according to the known kinetic mechanism of NagB_Ec. Assays were performed at 37°C and pH 7.4, conditions chosen to resemble the conditions of the bacterial cultures (Fig. 2, 3, and 6). The area bordered by a gray line indicates the range in GlcN6P and GlcNAc6P concentrations measured in vivo during growth on GlcN, where allosteric activation can be expected to be significant. The NagB_Ec concentration was 5 nM (hexamer). The data were fitted by nonlinear global regression analysis using the MWC equation for an exclusive-binding allosteric activator (25). The fitted MWC parameters are K_R(GlcN6P) = 0.85 ± 0.07 mM, K_A(GlcNAc6P) = 66 ± 17 μM, L₀ (MWC allosteric constant, defined by the T₀/R₀ ratio) = 5 × 10⁴, and c (defined as K_R/K_T) = 0.069 ± 0.005. These values can be compared to the previously measured values at 30°C and pH 7.7 [K_R(GlcN6P) = 0.55 + 0.05 mM, K_A(GlcNAc6P) = 35 ± 5 μM, L₀ = 10⁶, and c = 0.025 ± 0.002] (24, 26). Note that K_A(GlcNAc6P) is nearly 2-fold higher, and the parameter c, related to the free energy change of the T-R transition (K_R/K_T), is also 2-fold higher whereas L₀, the T/R ratio of the ligand-free enzyme, is 20-fold lower than at 30°C. (B) Fraction of NagB_Ec in the R conformer (R̄) and fractional saturation of the enzyme with substrate (ȳ_S) or allosteric activator (ȳ_A) during growth on GlcN and GlcNAc. R̄ was calculated with the parameters given in panel A using the following state equation: R̄ = [(1 + S/K_S)⁶]/{[L₀/(1 + A/K_A)⁶] (1 + cS/K_S)⁶ + (1 + S/K_S)⁶} (from reference , where S and A are the concentrations of substrate [GlcN6P] and allosteric activator [GlcNAc6P] and K_S and K_A are their microscopic dissociation constants) and the measured metabolite concentrations (Fig. 3; see Table S2 in the supplemental material). The fraction of the enzyme active sites occupied by GlcN6P (ȳ_S) is the v/V value (see above). The fraction of allosteric sites occupied by GlcNAc6P (ȳ_A) was calculated as [GlcNAc6P]/(K_A + [GlcNAc6P]). This is valid because only the R conformer of NagB_Ec binds the allosteric effector, GlcNAc6P.

FIG 5

(A) Kinetic properties of three GlcN6P deaminases, NagB_Ec, GamA, and NagB_Bs. The enzyme activity, expressed as v/V, of NagB_Ec, GamA, and NagB_Bs (and equivalent to the substrate saturation fraction [ȳ]), was measured in the absence (dotted lines) or presence (solid lines) of 1 mM GlcNAc6P at 37°C and pH 7.4. Data were fitted to the hyperbolic equation except for NagB_Ec in the absence of GlcNAc6P, which was fitted to the MWC equation for allosteric activation (2, 25). Enzyme concentrations were normalized to 40 nM per subunit. GlcNAc6P has no effect on the activities of the B. subtilis enzymes under the conditions used. (B) Kinetic parameters for the three enzymes in the presence of GlcNAc6P. Catalytic efficiency (also known as the specificity constant) was calculated as k_cat/K_m. Substrate inhibition was previously reported for NagB_Bs, when measured at pH 8.0 and at 22°C (8). Using our assay conditions (pH 7.4 and 37°C), we did not detect any inhibition by GlcN6P (up to 10 mM) or by GlcNAc6P (1 mM), but substrate inhibition was observed with our preparation when assayed under the conditions described previously (8).

FIG 6

(A) Growth rates of wild-type E. coli and E. coli with the nagB_Bs and gamA replacements on Glc, GlcN, and GlcNAc. Bacterial strains LAA199 (nagB_Ec⁺), LAA193 (gamA⁺), and LAA195 (nagB_Bs⁺) were grown in minimal MOPS medium at 37°C with 10 mM Glc, GlcN, or GlcNAc as the carbon source. Doubling times (DT) were calculated in exponential growth phase and are expressed in minutes ± the standard deviations for six biological replicate cultures. (Similar DT were measured for the strains carrying the nagB-lacZ fusion on a λ lysogen, MC-B1, MC-B362, and MC-B364.) (B) Transcriptional regulation by NagC of a nagB-lacZ fusion (on a λ lysogen) in wild-type E. coli and in E. coli with the nagB_Bs and gamA replacements, during growth on Glc, GlcN, and GlcNAc. MC-B1 (nagB_Ec⁺), MC-B362 (gamA⁺), and MC-B364 (nagB_Bs⁺) β-galactosidase activities (Miller units) were measured in minimal MOPS medium at 37°C with 10 mM Glc, GlcN, or GlcNAc as the carbon source.