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Literature summary for 1.4.1.3 extracted from

  • Tomita, T.; Kuzuyama, T.; Nishiyama, M.
    Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase (2011), J. Biol. Chem., 286, 37406-37413.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.4.1.3

Activating Compound

Activating Compound Comment Organism Structure
leucine the enzyme is subject to allosteric activation by leucine invlving ARg134, and Asp185, structural mechanism, overview Homo sapiens
leucine the enzyme is subject to allosteric activation by leucine, structural mechanism, overview Thermus thermophilus

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged GDH2 wild-type and mutants in Escherichia coli Homo sapiens
expression of His-tagged wild-type and mutant GDHA and GDHB in Escherichia coli strain BL21-CodonPlus (DE3)-RIL, and of untagged proteins Thermus thermophilus

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant untagged GDHA and GDHB in complex with each other and leucine, and GDHB in complex with glutamate, hanging drop vapor diffusion method using 0.1 M HEPES-NaOH, pH 7.0, and 0.6 M ammonium phosphate as the reservoir solution, X-ray diffraction structure determination and analysis at 2.6 A and 2.1 A resolution, respectively, molecular replacement Thermus thermophilus

Protein Variants

Protein Variants Comment Organism
A72D site-directed mutagenesis, the mutant shows no activation by leucine in contrast to the wild-type enzyme Thermus thermophilus
D166A site-directed mutagenesis, the mutant shows reduced activation by leucine compared to the wild-type enzyme Thermus thermophilus
D185A site-directed mutagenesis, the mutant shows no activation by leucine in contrast to the wild-type enzyme Homo sapiens
I71T site-directed mutagenesis, the mutant shows reduced activation by leucine compared to the wild-type enzyme Thermus thermophilus
R134A site-directed mutagenesis, the mutant shows no activation by leucine in contrast to the wild-type enzyme Thermus thermophilus
R151M site-directed mutagenesis, the mutant shows reduced activation by leucine compared to the wild-type enzyme Homo sapiens
Y38S site-directed mutagenesis, the mutant shows reduced activation by leucine compared to the wild-type enzyme Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate + H2O + NAD+ Thermus thermophilus
-
 2-oxoglutarate + NH3 + NADH + H+
-
 r
L-glutamate + H2O + NAD+ Homo sapiens
-
 2-oxoglutarate + NH3 + NADH + H+
-
 r
L-glutamate + H2O + NADP+ Thermus thermophilus
-
 2-oxoglutarate + NH3 + NADPH + H+
-
 r
L-glutamate + H2O + NADP+ Homo sapiens
-
 2-oxoglutarate + NH3 + NADPH + H+
-
 r

Organism

Organism UniProt Comment Textmining
Homo sapiens
-
-
-
Thermus thermophilus
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant GDH2 from Escherichia coli Homo sapiens
recombinant His-tagged wild-type and mutant GDHA and GDHB from Escherichia coli strain BL21-CodonPlus (DE3)-RIL by affinity chromatography and gel filtration, and of untagged proteins by heat treatment at 90°C for 20 min, hydrophobic interaction and anion exchange chromatography, and gel filtration to homogeneity Thermus thermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate + H2O + NAD+
-
 Thermus thermophilus 2-oxoglutarate + NH3 + NADH + H+
-
 r
L-glutamate + H2O + NAD+
-
 Homo sapiens 2-oxoglutarate + NH3 + NADH + H+
-
 r
L-glutamate + H2O + NADP+
-
 Thermus thermophilus 2-oxoglutarate + NH3 + NADPH + H+
-
 r
L-glutamate + H2O + NADP+
-
 Homo sapiens 2-oxoglutarate + NH3 + NADPH + H+
-
 r

Subunits

Subunits Comment Organism
dimer the GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex Thermus thermophilus
dimer the GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex Homo sapiens
hexamer the GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits Thermus thermophilus
hexamer the GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits Homo sapiens
More Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA, the regulatory subunit, and GdhB, the catalytic subunit, structure of the GdhA-GdhB-Leu complex, overview Thermus thermophilus
More Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA, the regulatory subunit, and GdhB, the catalytic subunit, structure of the GdhA-GdhB-Leu complex, overview Homo sapiens

Synonyms

Synonyms Comment Organism
GDH
-
 Thermus thermophilus
GDH
-
 Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Thermus thermophilus
7
-
 assay at Homo sapiens

Cofactor

Cofactor Comment Organism Structure
NAD+
-
 Thermus thermophilus
NAD+
-
 Homo sapiens
NADH
-
 Thermus thermophilus
NADH
-
 Homo sapiens
NADP+
-
 Thermus thermophilus
NADP+
-
 Homo sapiens
NADPH
-
 Thermus thermophilus
NADPH
-
 Homo sapiens

General Information

General Information Comment Organism
additional information glutamate is bound to the active site of GdhB, the GdhB-Glu complex takes an open-like structure. No substrate is found in the active site of the GdhAGdhB-Leu complex, while six leucine molecules are found at the interfaces of three subunits Thermus thermophilus