Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

Abstract

In orally acquired bacteria, the ability to counteract extreme acid stress (pH ⩽ 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved.

Keywords: Abs, absorbance; BmGadB, Brucella microti GadB; Brucella microti; Chloride activation; Cooperativity; EcGadB, Escherichia coli GadB; GABA, γ-aminobutyrate; GDAR, glutamate-dependent acid resistance; GadB, glutamate decarboxylase (B isoform); Glutamate decarboxylase; PLP, pyridoxal 5′-phosphate; Substituted aldamine; pH-dependent activity.

Graphical abstract

Fig. 1

Schematic representation of the role played by the major structural components of the E. coli GDAR system, the most extensively investigated AR system . l-glutamate (l-Glu, net charge 0) is taken up by the electrogenic l-Glu⁰/GABA⁺¹ antiporter GadC, an inner membrane protein. Decarboxylation of l-Glu via GadA/B consumes an intracellular H⁺ at each catalytic cycle, while GadC contributes to the generation of proton motive force by GABA (net charge +1) export. The structures of GadB at acidic pH (PDB: 1PMM) and GadC at pH 8.0 (PDB: 4DJK), the only one currently available, are shown as ribbon drawing generated with PyMol. The C-plug that in GadC is locking the substrate entry channel is shown in filled space.

Fig. 2

Clustal Omega (version 1.2.1) alignment of EcGadB and BmGadB. The residues in bold blue in BmGadB correspond to the N-terminal sequence experimentally determined by Edman degradation and on which numbering is based. The first 8 amino acids (in italics) in BmGadB are those reported in the NCBI database (Reference Sequence: YP_003105130.1), but absent in the protein sequence. The amino acid residues corresponding to the regions that in EcGadB undergo the most noticeable conformational changes are red. The residues participating in halide binding in EcGadB are in green background.

Fig. 3

Determination of molecular mass of BmGadB. Gel filtration chromatography of BmGadB (shown as red square on the calibration curve) on a Superdex 200 10/300 GL (GE Healthcare) column was carried out in 50 mM sodium acetate buffer, pH 4.5, containing 150 mM NaCl, at a flow rate of 0.5 ml/min. HMW Calibration Kit (GE Healthcare) included ferritin (440 kDa; a), aldolase (158 kDa; b), conalbumin (75 kDa; c), ovalbumin (44 kDa; d). 1 mg of BmGadB was loaded on the column immediately after calibration.

Fig. 4

pH-dependent absorbance changes. (A) Absorption spectra of BmGadB were recorded in 50 mM sodium acetate buffer at pH 3.5 (dark red), 4.2 (red), 4.5 (orange), 4.6 (magenta), 5.0 (violet), 5.4 (blue) and in 50 mM potassium phosphate buffer at pH 6.5 (green). Spectra were normalized taking into account the full PLP content (11.6 μM). The arrow indicates the direction of the change in absorbance at 420 nm upon pH increase. Inset: zoom of the 300–500 nm region. (B) The pH-dependent change in absorbance at 420 nm is represented in the absence (black squares) and presence (green squares) of 50 mM NaCl. The solid lines through the experimental points show the theoretical curves obtained using Hill equation as in GraphPad Prism 4.0.

Fig. 5

Effect of pH on the activity of BmGadB. Gad activity (Units mg⁻¹) was measured at 37 °C in 50 mM sodium acetate (pH 3.8–5.8) or phosphate (pH 6.0–6.5) buffer containing 40 μM PLP, 50 mM glutamate and in the absence (black circles) or presence (green circles) of 50 mM NaCl. The protein concentration was 0.5–2 μM. The solid lines through the experimental points represent the theoretical curves obtained using Hill equation to fit the data. The same experiment but with EcGadB is shown in the inset.

Fig. 6

Fluorescence emission spectra of BmGadB. Emission spectra were recorded at pH 4.0 (red line) and at pH 6.5 (green line) following excitation at 430 nm (A) and 295 nm (B). (C) Emission spectra of BmGadB (black line), EcGadB (blue line) and E. coli GadBH465A (violet line) were recorded following excitation at 345 nm at pH 6.5. The protein concentration of BmGadB was 13 μM. The protein concentration of EcGadB and GadBH465A was 3 μM. The buffers used were 50 mM sodium acetate at pH 4.0 and 50 mM potassium phosphate at pH 6.5.

Fig. 7

pH dependent cellular partition in EcGadB (left panel) and BmGadB (right panel). 12% SDS–PAGE of 30 μg-samples of cell supernatants (S), obtained after cell lysis, cytoplasmic (C) and membrane (M) fractions obtained as described in Section 2.4 at neutral (pH 7.2) and mildly acidic pH (pH 5.5 EcGadB or pH 5.1 BmGadB). The region of the gel encompassing the bands corresponding to EcGadB and BmGadB is shown with a blue box. Molecular weight (kDa) standards are shown on the right of the left panel. The decarboxylase activity is provided as percentage with respect to the corresponding starting activity in S (100%). The reported activity values represent the mean of 2–3 independent experiments, with a standard deviation not exceeding 10% of the stated value.