Biochemical characterization of a variant of Escherichia coli glutamate decarboxylase with improved GABA production at alkaline pH.

Background Glutamate decarboxylase (Gad), a structural component of the major acid resistance system in many orally-acquired, neutralophilic bacteria, yields CO2 and GABA, while consuming one proton/catalytic cycle [1]. Escherichia coli GadB (EcGadB) has a pH optimum in the pH range 4-5 and displays no activity at pH ≥ 6.5. The enzyme activity is therefore pH-controlled intracellularly. Based on the available crystal structures of EcGadB, the residues Asp86 and His465, highly conserved in bacterial Gads, were suggested to be major players in the control of EcGadB activity [1]. Objectives Bacterial Gad is regarded as an interesting tool for “green chemistry” applications because it can be employed to synthesize GABA, a non-proteinaceous amino acid, highly desirable in functional food and precursor of 2-pyrrolidone (an industrial solvent) for the synthesis of nylon 4 [2]. We engineered EcGadB at the level of residues Asp86 and His465 to generate a variant less sensitive to alkalinization. Methods EcGadB_Asp86Asn-His465Ala was overexpressed and purified. UV-visible and fluorescence spectrophotometry, circular dichroism, pH-dependent enzymatic activity assays and solvent kinetic isotope effect were employed to characterize this variant. Conclusions EcGadB_Asp86Asn-His465Ala, while retaining substrate specificity, acts as a more robust catalyst in the pH range 7-8 than wild type EcGadB and displays an altered solvent isotope effect. Because, pH is no longer a limiting reaction parameter for the mutant enzyme, we suggest that this variant can be a better tool for the industrial synthesis of GABA. [1] De Biase D, Pennacchietti E. (2012) Mol. Microbiol 86: 770-86. [2] Lammens TM et al. (2009) Green Chemistry 11: 1562-67.

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