The role of an active site aspartate residue in the catalytic activity of Glutamate decarboxylase from Escherichia coli.

Escherichia coli glutamate decarboxylase is a homohexameric PLP-dependent enzyme and a major structural component of the glutamate-based acid resistance system in this microorganism as well as in many orally-acquired, neutralophilic bacteria [1,2]. In fact the decarboxylation of L-glutamate, besides yielding γ-aminobutyrate (GABA) and CO2, consumes 1 H+/catalytic cycle, an activity shown to be beneficial for protecting the cell under extreme acid stress [1]. We have extensively characterized one of the two E. coli isoforms, the B isoform (EcGadB) and shown that it displays pH-dependency in activity in the acid range, being maximally active at pH 4-5 while showing negligible or no activity at or above pH 6.5. Based on the crystal structures of EcGadB at neutral and acidic pH, as well as in the presence of halides, and of a mutant form deleted at the N-terminal, we hypothesize that together with His465 (the penultimate residues in the amino acid sequence), Asp86 is a likely candidate for controlling the acidic range of activity of EcGad [3,4]. Notably, both residues are highly conserved in bacterial Gad [1]. The contribution to EcGadB spectroscopic and catalytic properties by His465, a critical residue for controlling active site access, was previously investigated [5]. In the present work, we carried out detailed biochemical characterization of the EcGadB-Asp86 mutant. However, in order to appreciate the contribution of Asp86 to the catalytic properties of EcGadB, it was necessary to incorporate the mutation Asp86→Asn in the mutant GadB_H465A, thereby avoiding the masking effect of His465 at pH>5.5. Our data show that, unlike wild-type EcGadB and GadB_H465A, the double mutant GadB_D86N¬-H465A, while retaining substrate specificity, is a more robust catalyst in the pH range 7-8 and displays an altered solvent kinetic isotope effect. Hence, GadB_D86N¬-H465A is less sensitive to pH increase during the decarboxylation reaction. We proposed that immobilization of EcGadB can be exploited for GABA synthesis at the industrial level [6]. GABA in turn can be used as precursor of 2-pyrrolidone, an industrial solvent, and of nylon 4. Thus mutant forms of EcGadB less sensitive to pH increase (i.e. > 5.5) are highly desirable. Based on our data, pH is no longer a limiting reaction parameter for GadB_D86N¬-H465A. References [1] De Biase D, Pennacchietti E. (2012) Mol. Microbiol 86: 770-86. [2] Lund P, Tramonti A, De Biase D. (2014) FEMS Microbiol Rev 38: 1091–125. [3] Capitani G, De Biase D, et al. (2003) EMBO J. 22: 4027-4037. [4] Gut H, Pennacchietti E, et al. (2006) EMBO J. 25: 2643-2651. [5] Pennacchietti E, Lammens TM, et al. (2009) J Biol Chem. 284: 31587-96. [6] Lammens TM, De Biase D, et al. (2009) Green Chemistry 11: 1562-67.

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