C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin.
Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADDeltaC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca2+/CaM, whereas in the presence of Ca2+/CaM all complexes shifted to approximately 680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC(500)), the ability to bind CaM in the presence of Ca2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca2+/CaM were completely abolished. Hence, responsiveness to Ca2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca2+/CaM.
Zik
Moriyah
M
Department of Life Sciences, Ben-Gurion University, POB 653, Beer-Sheva 84105, Israel.
Fridmann-Sirkis
Yael
Y
Fromm
Hillel
H
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000502:physiology*; D002147:Calmodulin / Q000737:chemistry / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry* / Q000502:physiology; D032306:Petunia / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
D002147:Calmodulin; D010446:Peptide Fragments; D010940:Plant Proteins; D005968:Glutamate Decarboxylase; D002118:Calcium