Escherichia coli glutamate decarboxylase (EcGad) is a homohexameric pyridoxal 5'-phosphate (PLP)-dependent enzyme. It is the structural component of the major acid resistance system that protects E. coli from strong acid stress (pH < 3), typically encountered in the mammalian gastrointestinal tract. In fact EcGad consumes one proton/catalytic cycle while yielding γ-aminobutyrate and carbon dioxide from the decarboxylation of l-glutamate. Two isoforms of Gad occur in E. coli (GadA and GadB) that are 99% identical in sequence. GadB is the most intensively investigated. Prompted by the observation that some transcriptomic and proteomic studies show EcGad to be expressed in conditions far from acidic, we investigated the structural organization of EcGadB in solution in the pH range 7.5-8.6. Small angle X-ray scattering, combined with size exclusion chromatography, and analytical ultracentrifugation analysis show that the compact and entangled EcGadB hexameric structure undergoes dissociation into dimers as pH alkalinizes. When PLP is not present, the dimeric species is the most abundant in solution, though evidence for the occurrence of a likely tetrameric species was also obtained. Trp fluorescence emission spectra as well as limited proteolysis studies suggest that PLP plays a key role in the acquisition of a folding necessary for the canonical catalytic activity.

Escherichia coli glutamate decarboxylase (EcGadB), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, is highly specific for L-glutamate and was demonstrated to be effectively immobilised for the production of γ-aminobutyric acid (GABA), its decarboxylation product. Herein we show that EcGadB quantitatively decarboxylates the L-isomer of D,L-2-amino-4-(hydroxyphosphinyl)butyric acid (D,L-Glu-γ-P H), a phosphinic analogue of glutamate containing C-P-H bonds. This yields 3-aminopropylphosphinic acid (GABA-P H), a known GABA B receptor agonist and provides previously unknown D-Glu-γ-P H , allowing us to demonstrate that L-Glu-γ-P H , but not D-Glu-γ-P H , is responsible for D,L-Glu-γ-P H antibacterial activity. Furthermore, using GABase, a preparation of GABA-transaminase and succinic semialdehyde dehydrogenase, we show that GABA-P H is converted to 3-(hydroxyphosphinyl)propionic acid (Succinate-P H). Hence, PLP-dependent and NADP +-dependent enzymes are herein shown to recognise and metabolise phosphinic compounds, leaving unaffected the P-H bond. We therefore suggest that the phosphinic group is a bioisostere of the carboxyl group and the metabolic transformations of phosphinic compounds may offer a ground for prodrug design.

Numerous commensal and pathogenic Gram-negative and Gram-positive bacteria are referred to as neutralophiles because they grow best at pH levels close to neutrality. Thus, exposure to harsh-to-mild acidic environments, such as those encountered in the digestive tract of animal hosts, in the phagosome of macrophages, in fermented foods, but also in the soil or in acid mine drainage, is a rather common encounter for neutralophiles during their life cycle. As a result, it is not surprising that most of them have evolved sophisticated molecular mechanisms to cope with low pH. These protective mechanisms provide neutralophiles with the ability to sense acid pH and keep under control the intracellular acidification of the cytoplasm, thus avoiding protons from reaching such harmful levels as to compromise cellular vitality, which relies on the proper functioning of many biological macromolecules at pH levels near neutrality.\ud \ud The aim of this chapter is to provide an overview of the most commonly employed, and best characterized, molecular systems in a number of Gram-positive and Gram-negative bacteria. How they work inside the cell and how their activity can be linked to virulence are highlighted. The biochemistry and distribution of the glutamate-dependent acid resistance system among orally acquired bacteria are described in some detail