Biochemical characterization of glutamic acid decarboxylase from Brucella microti

Glutamate decarboxylase (Gad; EC 4.1.1.15) is a pyridoxal 5´-phosphate (PLP)-dependent enzyme which catalyses the α-decarboxylation of L-glutamate (Glu) to γ-aminobutyrate (GABA) and CO2. Gad is the major structural component of the glutamate-dependent acid resistance (GDAR) system in several food-borne bacteria such as Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. The proton consuming activity of glutamate decarboxylase (GadB) and that of the cognate antiporter (GadC), which performs the Gluin/GABAout antiport, cooperate to protect bacteria from the extreme acid stress (pH < 2.5) encountered during the transit through the host stomach. Genome analysis indicated that the recently described Brucella microti, an environment-borne pathogenic Brucella species isolated from common vole, red fox and soil, possesses intact gadB and gadC genes. The role of GDAR system in B. microti acid resistance was recently investigated in light of a possible involvement in acid survival within the host. Indeed the GDAR system was recently shown to play an essential role in the resistance of B. microti to extreme acid shock and to contribute in the survival of the microorganism in a murine model following oral infection. Bacterial Gads share a set of strictly conserved residues. Several of these residues occupy critical positions in GadB from E. coli, the best characterized bacterial Gad. Thus the possibility exists that the pH-dependent molecular mechanism which controls the enzymatic activity of GadB from E. coli is conserved also in evolutionary distant bacterial species such as B. microti. The aim of this work was to investigate the molecular basis responsible for the intracellular activation of B. microti GadB. Overexpression, purification and biochemical characterization of GadB from B. microti are described and its spectroscopic and catalytic properties are analysed and compared with those of the E. coli enzyme.