SummaryFor successful colonization of the mammalian host, orally acquired bacteria must overcome the extreme acidic stress (pH < 2.5) encountered during transit through the host stomach. The glutamate-dependent acid resistance (GDAR) system is by far the most potent acid resistance system in commensal and pathogenic Escherichia coli, Shigella flexneri, Listeria monocytogenes and Lactococcus lactis. GDAR requires the activity of glutamate decarboxylase (GadB), an intracellular PLP-dependent enzyme which performs a proton-consuming decarboxylation reaction, and of the cognate antiporter (GadC), which performs the glutamate in/g-aminobutyrateout (GABA) electrogenic antiport. Herein we review recent findings on the structural determinants responsible for pH-dependent intracellular activation of E. coli GadB and GadC. A survey of genomes of bacteria (pathogenic and non-pathogenic), having in common the ability to colonize or to transit through the host gut, shows that the gadB and gadC genes frequently lie next or near each other. This gene arrangement is likely to be important to ensure timely co-regulation of the decarboxylase and the antiporter. Besides the involvement in acid resistance, GABA production and release were found to occur at very high levels in lactic acid bacteria originally isolated from traditionally fermented foods, supporting the evidence that GABA-enriched foods possess health-promoting properties.

Escherichia coli and other enterobacteria exploit the H þ -consuming reaction catalysed by glutamate decarboxylase to survive the stomach acidity before reaching the intestine. Here we show that chloride, extremely abundant in gastric secretions, is an allosteric activator producing a 10-fold increase in the decarboxylase activity at pH 5.6. Cooperativity and sensitivity to chloride were lost when the N-terminal 14 residues, involved in the formation of two triple-helix bundles, were deleted by mutagenesis. X-ray structures, obtained in the presence of the substrate analogue acetate, identified halide-binding sites at the base of each N-terminal helix, showed how halide binding is responsible for bundle stability and demonstrated that the interconversion between active and inactive forms of the enzyme is a stepwise process. We also discovered an entirely novel structure of the cofactor pyridoxal 5 0 -phosphate (aldamine) to be responsible for the reversibly inactivated enzyme. Our results link the entry of chloride ions, via the H þ /Cl À exchange activities of ClC-ec1, to the trigger of the acid stress response in the cell when the intracellular proton concentration has not yet reached fatal values.

Glutamate decarboxylase (GadB) from Escherichia coli is a hexameric, pyridoxal 5-phosphate-dependent enzyme catalyzing CO 2 release from the ␣-carboxyl group of L-glutamate to yield ␥-aminobutyrate. GadB exhibits an acidic pH optimum and undergoes a spectroscopically detectable and strongly cooperative pH-dependent conformational change involving at least six protons. Crystallographic studies showed that at mildly alkaline pH GadB is inactive because all active sites are locked by the C termini and that the 340 nm absorbance is an aldamine formed by the pyridoxal 5-phosphate-Lys 276 Schiff base with the distal nitrogen of His 465 , the penultimate residue in the GadB sequence. Herein we show that His 465 has a massive influence on the equilibrium between active and inactive forms, the former being favored when this residue is absent. His 465 contributes with n ≈ 2.5 to the overall cooperativity of the system. The residual cooperativity (n ≈ 3) is associated with the conformational changes still occurring at the N-terminal ends regardless of the mutation. His 465 , dispensable for the cooperativity that affects enzyme activity, is essential to include the conformational change of the N termini into the cooperativity of the whole system. In the absence of His 465 , a 330-nm absorbing species appears, with fluorescence emission spectra more complex than model compounds and consisting of two maxima at 390 and 510 nm. Because His 465 mutants are active at pH well above 5.7, they appear to be suitable for biotechnological applications.