What makes E. coli glutamate decarboxylase an amazing object for basic and biotech research

Glutamate decarboxylase (Gad; EC 4.1.1.15) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme which decarboxylates glutamate with concomitant proton consumption, CO2 release and GABA production. In Escherichia coli there are two Gad isoforms, GadA and GadB, which share 99.3% sequence similarity and are biochemically undistinguishable. Both isoforms have an acidic pH optimum, are hexamers and are major structural components to the glutamate-dependent acid resistance system, which protects pathogenic and commensal strains of E. coli from the extreme acid stress encountered during transit through the host stomach (1). In my laboratory and in collaboration with other groups (G. Capitani, PSI, Villigen Switzerland; M.C.R. Franssen, Wageningen University, The Netherlands) E. coli GadB is intensively studied. This enzyme undergoes spectroscopically detectable and strongly cooperative pH-dependent conformational change (2). The comparison of the enzyme’s crystal structures at pH 4.6 (active form) and 7.6 (inactive form) provided evidence that the pH change causes three major structural reorganizations (2). The first 15 residues of the N-terminal domain (residues 1-58) at acidic pH are required for recruitment of GadB to the membrane and for binding of chloride ions (2,3). The last 15 residues of the C-terminal small domain (residues 347-466) are ordered only at neutral pH, when they plug the active site funnel and form an entirely novel structure, an aldamine, with His465 side chain distal nitrogen making a reversible covalent bond with the PLP-Lys276 Schiff base (3). His465 has a massive influence on the equilibrium between active and inactive forms (4). Lastly, a β-hairpin (residue 300-313) in the large PLP-binding domain at neutral pH is required for fixing in place the C-terminal tail of a neighboring subunit (3), whereas at acidic pH provides residues that partecipate in catalysis. E. coli GadB was also efficiently immobilised in calcium alginate beads (5). The industrial production of GABA from glutamic acid, which is abundant in waste streams from biofuel production, is regarded as an interesting alternative for the synthesis of nitrogen-containing bulk chemicals, thereby decreasing the dependency on fossil fuels (5). We designed and characterized GadB site specific mutants with a range of activity significantly extended toward alkaline pH, to be employed for the above applications.