Functional Roles of the Hexamer Organization of Plant Glutamate Decarboxylase

Glutamate decarboxylase (GAD) is a pyridoxal 5´-phosphate (PLP)-dependent enzyme that catalyzes the irreversible α-decarboxylation of glutamate to γ-aminobutyrate. The enzyme is widely distributed in eukaryotes and prokaryotes, although its function varies in different organisms. A unique feature of plant GAD is the presence of a calmodulin (CaM)-binding domain at its C-terminus. In plants, transient elevation of cytosolic Ca2+ in response to different types of stress is thought to be responsible for GAD activation via CaM. The crystal structure of GAD1 from Arabidopsis thaliana shows that the enzyme is a hexamer composed of trimer-of-dimers. Herein, we show that in solution GAD1 exists as a dimer/hexamer equilibrium mixture, and we estimate the dissociation constant (Kd) for the hexamer under different conditions. The association of dimers into hexamers is promoted by a number of conditions, including high protein concentrations and low pH. Notably, binding of Ca2+/CaM abolishes GAD1 oligomer dissociation by forming a stable complex in which three CaM bind to a GAD1 hexamer. The GAD1 N-terminal domain is critical for maintaining the oligomeric state, since the removal of the first 24 N-terminal residues dramatically affects the oligomerization process by producing an enzyme that exists only as a dimer. The deleted mutant retains decarboxylase activity, highlighting the dimeric nature of the basic structural unit of GAD1. Site-directed mutagenesis identified a hexamerization ‘hot spot’ centered on Arg24 in the N-terminal domain. Mutation of this critical Arg residue to Ala prevents hexamer formation in solution. Surprisingly, both the dimeric ArgAla and 1-24 mutant enzymes form a stable hexamer in the presence of Ca2+/CaM. The present data, clearly revealing that the GAD1 oligomeric state is highly responsive to a number of experimental parameters, might have functional relevance in vivo and is discussed in the light of the biphasic regulation of GAD1 activity by pH and Ca2+/CaM in plant cells.