Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase

Abstract

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.

Fig. 1. Surface (top) and cartoon (bottom) representation of the GadB hexamer at neutral pH (A) and low pH (B). Figures were prepared with MSMS (Sanner et al., 1996), DINO (www.dino3d.org), MOLSCRIPT (Kraulis, 1991) and RASTER3D (Merritt and Murphy, 1997).

Fig. 2. Superposition of the three N-termini from one layer of the GadB hexamer at neutral pH (cyan and atom colors) and at low pH (yellow and atom colors). The N-termini for the low-pH form are labelled.

Fig. 3. Superposition of the GadB (cyan, neutral-pH form) and DDC (red) Cα traces. N- and C- termini are labelled, as are the DDC residues (327 and 340) flanking the region for which no electron density is visible.

Fig. 4. Effect of pH on the cellular localization of GadB wt (left panels) and GadBΔ1–14 (right panels). Upper panels: 10% SDS–PAGE of cell supernatants (S, 20 µg), obtained after cell lysis and centrifugation to remove cell debris; the cytoplasmic (C, 20 µg) and membrane (M₁, 20 µg) fractions were obtained as described in Materials and methods. For GadB wt, an additional lane (M₂) represents the 1% lauroyl sarcosine-solubilized membrane fraction after the last ultracentrifugation, still containing some GadB wt. Molecular weight (kDa) standards are shown in the middle of the right panel and on the left hand side of the left panel. The vertical bar between panels refers to the anti-GadB probed region. Lower panels: immunoblot analysis of samples as in upper panel, probed with anti-GadB polyclonal antibodies. Due to the different amount of GadB (wt or mutant) present in the cell supernatant (S) the loadings were 2.5–2.8 µg for GadB wt and 8.4–8.9 µg for GadBΔ1–14. The C, M₁ and M₂ fractions were loaded according to the total protein partition between these fractions, with respect to S. The decarboxylase activity is in total units (U × 10³, upper line) and as a percentage (%, lower line) with respect to the cell supernatant (S = 100%). The values with an asterisk refer to the decarboxylase activity present in the resuspended membrane fraction before separation from the insoluble material, thus allowing measurement of the enzyme still present in the M₂ fraction. The reported activity values represent the means of three independent experiments, with a variation that does not exceed 10% of the stated value.

Fig. 5. Effect of pH on the absorbance spectra of GadBΔ1–14. Spectra of GadB (6.9 µM) were determined in 0.1 M Na acetate in the pH range 3–7.15. Spectra at pH 3.20, 5.05, 5.25, 5.34, 5.47, 5.55, 5.88 and 7.15 are shown. In the inset, the pH variation at 418 nm is represented. The solid line is that of best fit to equation 1 (Materials and methods), with pK = 5.46 ± 0.01, Abs_HnE = 0.0604 ± 0.0005, Abs_E = 0.0095 ± 0.0007 and n = 3.0 ± 0.2. Curve fitting obtained by using the 338-nm absorbance readings gave similar pK and n values (pK = 5.45 ± 0.017; n = 2.4 ± 0.2).

Fig. 6. Active site views. (A) Active sites of GadB (neutral pH, grey and atom colours) and DDC (yellow and atom colours) superimposed. For clarity, no water molecules are shown. Asterisks indicate the two Cys residues of GadB (Cys130 and Cys165), which may form a disulfide bridge. (B) Active site of GadB at low pH. The final 2mF_o – DF_c electron density for PLP, Lys276 and the bound acetate is contoured in green at 1σ level. (C) Model for the external aldimine intermediate with glutamate. Relevant hydrogen bonds appear as green dotted lines, bound water molecules as red spheres.