title: 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.
authors: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.
authors: Gaia
authors: G
authors: Grassini
authors: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.
authors: Eugenia
authors: E
authors: Pennacchietti
authors: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.
authors: Francesca
authors: F
authors: Cappadocio
authors: Université de Montpellier, Centre d'études d'agents Pathogènes et Biotechnologie pour la Santé (CPBS), F-34293 Montpellier, France ; CNRS, FRE 3689, CPBS, F-34293 Montpellier, France.
authors: Alessandra
authors: A
authors: Occhialini
authors: Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, 04100 Latina, Italy.
authors: Daniela
authors: D
authors: De Biase
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