Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana - characterization of the enzyme's in vitro truncation by thiol endopeptidase activity
Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin activated cytosolic enzyme that produces {gamma}-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 that in vivo phosphorylation 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 plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 following its expression in Escherichia coli to facilitate studies of the impact of site-specific phosphorylation on its kinetic properties. However, in vitro proteolytic 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 {degrees}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 an E. coli cysteine 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 ({micro}mol 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.