Biochemical and biophysical characterization of Glutamate decarboxylase of bacterial origin

Bacteria are constantly exposed to harsh conditions during their life cycle. Thus, the development of molecular strategies to overcome stressful environments is required for their survival. The acid stress is considered one of the most severe life-threatening conditions for neutralophiles, i.e. microorganisms that live and replicate best at neutral pH. An acidic stress is frequently encountered in natural habitats and/or within the host during the infectious process. Therefore, if not properly counteracted, it can lead to death. In food-borne neutralophilic bacteria, the acid stress in mostly encountered during their transit through the host gastro-intestinal tract where the environmental pH fluctuates between 1.5-2.5 in the stomach and 4.5-5.0 in the distal intestine. In the last decades, several acid resistance molecular mechanisms have been identified; amongst them, the glutamate-dependent acid resistance mechanism is regarded the most potent. In Escherichia coli it relies on pyridoxal 5’-phosphate (PLP)-dependent glutamate decarboxylase GadA/B, which catalyze the proton-consuming decarboxylation of glutamate (L-Glu) to γ-aminobutyric acid (GABA), which is then exported by GadC, a Glu/GABA antiporter. The reaction consumes 1 H+/cycle. The pH-dependent biochemical properties of GadB from E. coli (EcGadB) have been studied in depth since the first publication of the purification protocol (De Biase et al., 1996), but several unanswered questions remain. As part of the research work leading to my PhD degree, I investigated EcGadB properties at different biochemical and biophysical levels, with regard to: i) the influence of PLP and of exposure to neutral-alkaline pH values on the quaternary structure of EcGadB; ii) the role of aspartate 86 in the pH-dependent control of EcGadB enzymatic activity. Moreover, I carried out a preliminary biochemical characterization of GadB from Mycobacterium tuberculosis to shed light on the hypothetical involvement of a Gad enzyme on either acid resistance within phagosome/phagolysosome in macrophages or as a key enzyme of the GABA-shunt, which provides succinate from α-ketoglutarate, thus refilling the otherwise incomplete TCA cycle. Finally, I focused on the antimicrobial activity of analogues of dicarboxylic acids, the results of which are under final assessment for deposit as an Italian patent.