Heterologous expression and purification of glutamate decarboxylase-1 from the model plantArabidopsis thaliana: characterization of the enzyme’sin vitrotruncation by thiol endopeptidase activity

Abstract

ABSTRACTPlant glutamate decarboxylase (GAD) is a Ca2+-calmodulin activated cytosolic enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. This pathway circumvents the 2-oxoglutarate to succinate reactions of the mitochondrial tricarboxylic acid cycle. Our prior research established thatin vivophosphorylation of the root-specific AtGAD1 isozyme (AT5G17330) occurs at multiple N-terminal serine residues, following Pi resupply to Pi-starved cell cultures of the model plantArabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 following its expression inEscherichia colito facilitate studies of the impact of site-specific phosphorylation on its kinetic properties. However,in vitroproteolytic truncation of a 5 kDa polypeptide from the C-terminus of 59 kDa AtGAD1 subunits occurred during its purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing partial AtGAD1 proteolysis during incubation of clarified extracts at 23 °C. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated AtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalytic activity, and that AtGAD1 is susceptible to partial degradation either by anE. colicysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/calmodulin in extraction and chromatography buffers facilitated the purification of non-proteolyzed AtGAD1 to a specific activity of 27 (µmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed His6-AtGAD1 was >40% less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent unwanted proteolysis before conducting kinetic studies of purified regulatory enzymes.