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Literature summary for 6.3.1.2 extracted from

  • Ferreira, M.J.; Vale, D.; Cunha, L.; Melo, P.
    Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula crucial for enzyme activity and needless for protein import into the plastids (2017), Plant Physiol. Biochem., 111, 226-233 .
    View publication on PubMed
Search for more literature for 6.3.1.2

Cloned(Commentary)

Cloned (Comment) Organism
isozyme GS2a, DNA and amino acid sequence determination and analysis, sequence comparisons of GS isoforms, recombinant expression of His6-tagged isozyme GS2a in Escherichia coli strain BL21(DE3) Codon Plus, transient expression of the isozyme GS2a in Nicotiana tabacum cv. Petit Havana SR1 foliar cells via transfection with Agrobacterium tumefaciens strain GV3101 Medicago truncatula

Protein Variants

Protein Variants Comment Organism
additional information generation of a construct, MtGS2DELTACT, encoding MtGS2a mature protein (without the N-terminal plastid targeting signal) truncated at the C-terminal (lacking the last 16 amino acid residues) and with a hexahistidine tag (His-tag) fused to the N-terminal in the pET28a expression vector. The solubility and the thermal stability of MtGS2a proteins with and without the C-terminal extension peptide are similar. The specific activity of the recombinant protein without the C-terminal extension peptide, either transferase or syntethase activity, is lower compared with that of the protein with the C-terminal extension peptide Medicago truncatula

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information the enzymatic rate of GS enzymes shows a hyperbolic curve typical of the enzymes that follow the Michaelis-Menten equation regarding the ATP and glutamate and a sigmoidal curve relative to the hydroxylamine concentration Medicago truncatula
0.85
-
 hydroxylamine pH not specified in the publication, 30°C, mutant mature enzyme lacking the C-terminal peptide Medicago truncatula
0.92
-
 hydroxylamine pH not specified in the publication, 30°C, wild-type mature enzyme Medicago truncatula
1.83
-
 ATP pH not specified in the publication, 30°C, wild-type mature enzyme Medicago truncatula
1.95
-
 ATP pH not specified in the publication, 30°C, mutant mature enzyme lacking the C-terminal peptide Medicago truncatula
5.7
-
 L-glutamate pH not specified in the publication, 30°C, wild-type mature enzyme Medicago truncatula
11.52
-
 L-glutamate pH not specified in the publication, 30°C, mutant mature enzyme lacking the C-terminal peptide Medicago truncatula

Localization

Localization Comment Organism GeneOntology No. Textmining
chloroplast the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids. The C-terminal extension peptide of plastid-located glutamine synthetase isozyme GS2a from Medicago truncatula is crucial for enzyme activity but needless for protein import into the plastids. The 16 amino acid C-terminal extension is present in all known plastid GS proteins Medicago truncatula 9507
-

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + L-glutamate + NH3 Medicago truncatula
-
 ADP + phosphate + L-glutamine
-
 ?

Organism

Organism UniProt Comment Textmining
Medicago truncatula O04998
-
-

Posttranslational Modification

Posttranslational Modification Comment Organism
proteolytic modification the first 49 amino acids of the N-terminus are predicted to be the transit peptide, the sorting signal for targeting nucleus-encoded proteins to the plastids, which is cleaved during the import. The protein can only assemble to its active form when this sorting signal is removed Medicago truncatula

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged isozyme GS2a from Escherichia coli strain BL21(DE3) Codon Plus by m´nickel affinity chromatography Medicago truncatula

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-Glu + hydroxylamine
-
 Medicago truncatula ADP + phosphate + gamma-glutamyl hydroxamate
-
 ?
ATP + L-glutamate + NH3
-
 Medicago truncatula ADP + phosphate + L-glutamine
-
 ?

Subunits

Subunits Comment Organism
? x * 47000, about, full-length wild-type isozyme GS2a, sequence calculation, x * 41700, about, mature and active isozyme GS2a without transit peptide, sequence calculation, x * 40000, about, mutant isozyme GS2a with cleaved N- and C-terminal peptides, sequence calculation Medicago truncatula

Synonyms

Synonyms Comment Organism
GS2a
-
 Medicago truncatula

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Medicago truncatula

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Medicago truncatula

General Information

General Information Comment Organism
evolution glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species Medicago truncatula
metabolism glutamine synthetase is a key enzyme in plant nitrogen metabolism Medicago truncatula
additional information the C-terminal extension peptide of plastid-located glutamine synthetase from Medicago truncatula is crucial for enzyme activity but needless for protein import into the plastids. The C-terminal extension peptide does not affect the solubility or the stability of the protein but likely the interaction of the enzyme with its substrates. The first 49 amino acids of the N-terminus are predicted to be the transit peptide, the sorting signal for targeting nucleus-encoded proteins to the plastids, which is cleaved during the import Medicago truncatula