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Literature summary for 4.1.1.15 extracted from

  • Astegno, A.; Capitani, G.; Dominici, P.
    Functional roles of the hexamer organization of plant glutamate decarboxylase (2015), Biochim. Biophys. Acta, 1854, 1229-1237 .
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene gad1, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)pLysS Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme, X-ray diffraction structure determination and analysis Arabidopsis thaliana

Protein Variants

Protein Variants Comment Organism
additional information removal of the first 24 N-terminal residues of AtGAD1 dramatically affects oligomerization by producing a dimeric enzyme. The deleted mutant retains decarboxylase activity, highlighting the dimeric nature of the basic structural unit of AtGAD1. The dimeric mutant enzyme forms a stable hexamer in the presence of Ca2+/CaM1. Binding of Ca2+/CaM1 appears to restore the hexamer species, since the gel filtration profiles of the mutant AtGAD1-DELTA1-24-Ca2+/CaM1 complex shows the same elution volume of the AtGAD1-Ca2+/CaM1 complex across the entire pH range. The AtGAD1-DELTA1-24 enzyme shows decreased thermal stability compared with the wild-type form Arabidopsis thaliana
R24A site-directed mutagenesis of key residue Arg24 in the N-terminal domain to Ala prevents hexamer formation of enzyme AtGAD1 in solution. The dimeric mutant enzyme forms a stable hexamer in the presence of Ca2+/ CaM1 Arabidopsis thaliana

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ in plants, transient elevation of cytosolic Ca2+ in response to different types of stress is responsible for GAD activation via calmodulin. Binding of Ca2+/CaM1 abolishes the dissociation of the AtGAD1 oligomer Arabidopsis thaliana

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
105000
-
homodimeric enzyme, gel filtration and native PAGE Arabidopsis thaliana
110000
-
truncated mutant enzyme AtGAD1-DELTA1-24, gel filtration Arabidopsis thaliana
342000
-
homohexameric enzyme, gel filtration and native PAGE Arabidopsis thaliana

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate Arabidopsis thaliana
-
4-aminobutanoate + CO2
-
?

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana Q42521
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)pLysS Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate
-
Arabidopsis thaliana 4-aminobutanoate + CO2
-
?

Subunits

Subunits Comment Organism
homodimer the basic structural unit of AtGAD1 is a homodimer Arabidopsis thaliana
homohexamer hexamer composed of a trimer of dimers. Hexamerization strongly contributes to the stability of the enzyme Arabidopsis thaliana
More in solution AtGAD1 is in a dimer-hexamer equilibrium. Binding of Ca2+/CaM1 abolishes the dissociation of the AtGAD1 oligomer. The AtGAD1N-terminal domain is critical for maintaining the oligomeric state. Arg24 in the N-terminal domain is a key residue. The oligomeric state of AtGAD1 is highly responsive to a number of experimental parameters and may have functional relevance in vivo in the light of the biphasic regulation of AtGAD1 activity by pH and Ca2+/CaM1 in plant cells. Tryptic peptide mapping. Effect of pH on the dissociation of hexameric AtGAD1 in the pH range 6.0-8.0, overview. A flexible and exposed stretch spanning residues 1-24 is the minimum region required for assembly of hexamer Arabidopsis thaliana

Synonyms

Synonyms Comment Organism
AtGAD1
-
Arabidopsis thaliana

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Arabidopsis thaliana

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
56
-
truncated enzyme mutant AtGAD1-DELTA1-24, T50 value Arabidopsis thaliana
70
-
wild-type enzyme, T50 value Arabidopsis thaliana

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.8
-
assay at Arabidopsis thaliana

Cofactor

Cofactor Comment Organism Structure
Calmodulin 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 responsible for GAD activation via CaM Arabidopsis thaliana
pyridoxal 5'-phosphate
-
Arabidopsis thaliana

General Information

General Information Comment Organism
evolution the enzyme belongs to the fold type I family of PLP-enzymes Arabidopsis thaliana
additional information hexamerization strongly contributes to the stability of the enzyme. Plant GADs possess four conserved basic residues in their first 24 N-terminal amino acid region (H5,H15, R21, and R24 in AtGAD1). Two of the four residues (H15 and R24) are located at the interfaces between dimeric units Arabidopsis thaliana
physiological function compared to GADs from other organisms, plant GADs possess a unique feature, namely, the presence of a C-terminal calmodulin binding site (CaMBD). This characteristic confers plant GADs an additional regulatory mechanism by making them responsive to cytosolic calcium (Ca2+), thus revealing that at least two mechanisms exist, by which GAD activity can be stimulated in vitro and in vivo, namely, acidic pH and Ca2+/CaM. Transient elevation of cytosolic Ca2+ in response to different types of stress is responsible for GAD activation via CaM Arabidopsis thaliana