Biochemical and structural characterization of calcium/calmodulin dependent glutamate decarboxylase 1 from Arabidopsis thaliana

Glutamate decarboxylase (GAD) is a pyridoxal 5´-phosphate (PLP)-dependent enzyme that catalyzes glutamate to γ-aminobutyrate (GABA) conversion. With respect to GADs from other organisms, plant GADs possess a unique feature, namely the presence of a C-terminal calmodulin (CaM) binding site. As a consequence, plant GADs exhibit dual regulation by pH, with maximum activity at pH 5.8 and by calcium (Ca2+)/CaM activation at neutral pH, nearly negligible at acidic pH. Herein, a detailed biochemical and structural description of the Arabidopsis thaliana enzyme GAD1 and of GAD1-CaM1 complex has been performed. A combination of UV/Vis spectrophotometry and fluorescence spectroscopy provided significant information on GAD1 active site and of its changes upon CaM1 binding. Importantly, mutagenesis studies allowed the identification of the key residues in the C-terminal region of GAD1 for regulation by calmodulin and by pH. Further, the crystal structure of Arabidopsis thaliana GAD1 in its CaM-free state at pH close to 6, where the enzyme reaches maximal turnover in the absence of CaM, was determinated. The protein is a 342-kDa homohexamer composed of a trimer of dimers, consisting of two layers of three subunits each, where the dimers contribute one subunit to each layer. A low-resolution structure of the calmodulin-activated GAD1 complex by Small-Angle X-ray Scattering (SAXS) was also obtained in order to elucidate the structure of the complex and the mode of activation. CaM1 activates GAD1 in a unique way by relieving two C-terminal autoinhibition domains of adjacent active sites, forming a 393 kDa GAD1-CaM1 complex with an unusual 1:3 stoichiometry, thus representing a prototype for a novel CaM target interaction mode. As an effort to elucidate GAD1 quaternary structure in solution, its dissociation/association and conformational properties were investigated by using an array of chromatographic and spectroscopic techniques. Size exclusion chromatography (SEC) and native PAGE clearly showed that GAD1 protein exists in a hexamer-dimer equilibrium in solution which is dependent on some specific conditions such as protein concentration, pH and NaCl concentration. The dissociation constant (Kd) for the hexamer under these different conditions was estimated according to the Manning method [Manning et al., 1996], adapted to the hexamer-dimer equilibrium. The strong influence of pH on GAD1 ability to associate suggested that electrostatic forces mediate hexamer formation. Inspection of GAD1 hexameric structure revealed the main contacts between the three dimers are set up by reciprocal salt bridges between His15 and Glu338. To investigate the importance of this ion pair contacts, H15LE338A double mutant was generated and biochemically characterized. Surprisingly, the mutant showed a Kd value strongly decreased, at least 50-fold lower than that of wild type (wt), with higher thermal stability and higher catalytic activity. The 3D structure of GAD1 indicates that N-terminal domain plays an important role in formation and stabilization of the hexamer. The truncation of GAD1 by removing the first 24 amino acid of the N-terminal domain resulted in the complete dimerization of the enzyme, confirming the N-terminal domain structural role in oligomerization. The deleted mutant retains decarboxylase activity, which is further evidence for the dimeric nature of the basic structural unit of GAD1. Interestingly Ca2+/CaM-binding essentially abolishes both protein concentration and pH dissociation dependence of GAD1 wt and of all its mutants forming and/or stabilizing a complex with a molecular weight in agreement with the 1:3 GAD1-CaM1 stoichiometry determinated by a SAXS solution study. An interpretation of the biological significance of the two coexisting regulation mechanisms of GAD1 was also proposed. Thanks to its two levels of regulation, GAD1 can respond flexibly to different kinds of cellular stress occurring at different pH values.