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Literature summary for 1.4.1.4 extracted from

  • Sharkey, M.A.; Oliveira, T.F.; Engel, P.C.; Khan, A.R.
    Structure of NADP(+) -dependent glutamate dehydrogenase from Escherichia coli - reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases (2013), FEBS J., 280, 4681-4692.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
sequence comparisons, expression of wild-type and mutant enzymes Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
purified GDH in the absence of reactants, hanging drop vapour diffusion method, mixing of 10 mg/ml protein in 20 mM Tris-HCl, pH 7.6, with 15-25% PEG 3350, Tris/HEPES, pH 70-8.0, and 0.2 M NaCl, 18°C, 2 days, X-ray diffraction structure determination and analysis at 2.5 A resolution, modelling Escherichia coli

Protein Variants

Protein Variants Comment Organism
K286Q site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme Escherichia coli
K286Q/R289Q/R292Q site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme Escherichia coli
K286Q/R289Q/R292Q/S264L site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme Escherichia coli
K286Q/R289Q/R292Q/S264L/S240A site-directed mutagenesis, the mutant shows highly increased KM for NADP+ compared to the wild-type enzyme Escherichia coli
R289Q site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme Escherichia coli
R292Q site-directed mutagenesis, the mutant shows increased KM for NADP+ compared to the wild-type enzyme Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0184
-
NADP+ wild-type enzyme, pH 8.0, temperature not specified in the publication Escherichia coli
0.0328
-
NADP+ mutant K289Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.0819
-
NADP+ mutant K292Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.129
-
NADP+ mutant K286Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.929
-
NADP+ mutant K286Q/R289Q/R292Q , pH 8.0, temperature not specified in the publication Escherichia coli
18.3
-
NADP+ mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication Escherichia coli
19.06
-
NADP+ mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate + H2O + NADP+ Escherichia coli
-
2-oxoglutarate + NH3 + NADPH + H+
-
r

Organism

Organism UniProt Comment Textmining
Escherichia coli P00370
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate + H2O + NADP+
-
Escherichia coli 2-oxoglutarate + NH3 + NADPH + H+
-
r

Subunits

Subunits Comment Organism
hexamer EcGDH is a hexamer with 32 symmetry, an assembly. Protomer F (EcGDH-F) is the only molecule in the hexamer that adopts an open conformation that is sufficiently wide for diffusion of both substrate and cofactor, structure comparison to the enzyme from Clostridium symbiosum and other GDHs, overview Escherichia coli
More modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring alpha-helical hairpin to phosphate recognition, sequence-structure relationship, overview Escherichia coli

Synonyms

Synonyms Comment Organism
NADP+-dependent GDH
-
Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.5
-
NADP+ mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication Escherichia coli
6.9
-
NADP+ mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication Escherichia coli
24
-
NADP+ mutant K286Q/R289Q/R292Q, pH 8.0, temperature not specified in the publication Escherichia coli
34.2
-
NADP+ mutant K286Q, pH 8.0, temperature not specified in the publication Escherichia coli
37.3
-
NADP+ wild-type enzyme, pH 8.0, temperature not specified in the publication Escherichia coli
37.6
-
NADP+ mutant K289Q, pH 8.0, temperature not specified in the publication Escherichia coli
47.3
-
NADP+ mutant K292Q, pH 8.0, temperature not specified in the publication Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
NADP+ binding structure involving Asp263 and Ser264, residues P7 and P8, molecular modelling of NADP+ onto the free enzyme structure, overview Escherichia coli
NADPH
-
Escherichia coli

General Information

General Information Comment Organism
additional information modelling of NADP+ in domain II reveals the potential contribution of positively charged residues from a neighbouring alpha-helical hairpin to phosphate recognition, sequence-structure relationship, overview. A single sequence accommodates both coenzymes in the dual-specificity GDHs of animals Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.00000013
-
NADP+ mutant K286Q/R289Q/R292Q/S264L, pH 8.0, temperature not specified in the publication Escherichia coli
0.00000038
-
NADP+ mutant K286Q/R289Q/R292Q/S264L/S240A, pH 8.0, temperature not specified in the publication Escherichia coli
0.0000258
-
NADP+ mutant K286Q/R289Q/R292Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.000115
-
NADP+ mutant K289Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.000265
-
NADP+ mutant K286Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.000578
-
NADP+ mutant K292Q, pH 8.0, temperature not specified in the publication Escherichia coli
0.00203
-
NADP+ wild-type enzyme, pH 8.0, temperature not specified in the publication Escherichia coli