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Literature summary for 1.4.1.3 extracted from

  • Tomita, T.; Matsushita, H.; Yoshida, A.; Kosono, S.; Yoshida, M.; Kuzuyama, T.; Nishiyama, M.
    Glutamate dehydrogenase from Thermus thermophilus is activated by AMP and leucine as a complex with catalytically inactive adenine phosphoribosyltransferase homolog (2019), J. Bacteriol., 201, e00710-18 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.4.1.3

Activating Compound

Activating Compound Comment Organism Structure
AMP AMP activated GDH activity of the ternary complex, but not the GdhA-GdhB binary complex. APRTh mediates the allosteric activation of GDH by AMP. Reductive amination and oxidative deamination activity is enhanced by up to 6.3 and 2.5fold, respectively, by the addition of 1 mM AMP. Thermus thermophilus
APRTh TTC1249 (APRTh), a 23-30 kDa protein, which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, forms a ternary complex with the enzyme heterodimer, heterocomplex formation and structure, interaction analysis and function, detailed overview. APRTh mediates the allosteric activation of GDH by AMP Thermus thermophilus
L-leucine the crystal structure of GdhA/GdhB in a complex with leucine and biochemical analysis reveals that leucine is bound to the pockets at the GdhA-GdhA, GdhA-GdhB, and GdhB-GdhB interfaces. leucine binding increases the turnover of the GDH reaction, possibly through acceleration of the open-close cycle of the active-site cleft between the catalytic domain and NAD-binding domains Thermus thermophilus
additional information in reductive amination, the influence of AMP and leucine on the Km for NADH, 2-oxoglutarate, and ammonium is not large, although approximately 4.5, 5.3, and 10.2fold increases in Km for 2-OG are observed with AMP, leucine, and the copresence of AMP and leucine, respectively. Activation profiles of GDH activity of the TtGDH-APRTh complex by leucine and AMP, overview Thermus thermophilus

Cloned(Commentary)

Cloned (Comment) Organism
two GDH homologues, gdhA and gdhB, with 46% amino acid sequence identity, are present in a tandem manner as genes TTC1212 and TTC1211 on the genome of Thermus thermophilus, recombinant expression of the His-tagged ternary complex TtGDH-APRTh in Escherichia coli Thermus thermophilus

Protein Variants

Protein Variants Comment Organism
additional information growth analysis of the aprth knockout strain (Tt27DELTAAPRTh) and aprth-overexpressing strain (Tt27NStHisAPRTh) of Thermus thermophilus in minimal medium. The Tt27DELTAAPRTh strain exhibits delayed growth and requires approximately 36 h to reach the early stationary phase, whereas the wild-type strain reaches this phase after 21 h of cultivation. The overexpressing Tt27NStHisAPRTh strain exhibits better growth than even the wild-type strain Thermus thermophilus

Inhibitors

Inhibitors Comment Organism Structure
GTP
-
 Thermus thermophilus
NADH allosteric enzyme regulation Thermus thermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.003
-
 NADH recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
0.0051
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.0053
-
 NADH recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
0.0058
-
 NADH recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
0.008
-
 NADH recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.0229
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
0.024
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.027
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
0.052
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
0.056
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
0.13
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
0.16
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
0.27
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, low Glu concentration, with AMP, pH and temperature not specified in the publication Thermus thermophilus
0.45
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
0.77
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
1.2
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, low Glu concentration, pH and temperature not specified in the publication Thermus thermophilus
19
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, high Glu concentration, with AMP, pH and temperature not specified in the publication Thermus thermophilus
29
-
 NH3 recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
33
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
43
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
46
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
52
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, high Glu concentration, pH and temperature not specified in the publication Thermus thermophilus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
315000
-
 recombinant His-tagged binary complex TtGDH, gel filtration Thermus thermophilus
331000
-
 recombinant His-tagged ternary complex TtGDH-APRTh, gel filtration Thermus thermophilus

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Thermus thermophilus Q93IM2 subunit GdhA
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged ternary complex TtGDH-APRTh from Escherichia coli by nickel affinity chromatography and gel filtration Thermus thermophilus

Substrates and Products (Substrate)

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

Subunits

Subunits Comment Organism
heterohexamer 3 * 46328, GdhA, + 3 * 46112, GdhB, sequence calculation and SDS-PAGE Thermus thermophilus
More GDH is composed of two heterologous subunits, GdhA and GdhB. GdhA and GdhB form a heterohexamer, in which GdhB acts as the catalytic subunit and GdhA acts as the regulatory subunit to sense leucine. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, forms a ternary complex with the enzyme heterodimer. The ternary complex exhibits GDH activity that is activated by leucine, as observed for the GdhA-GdhB binary complex Thermus thermophilus

Synonyms

Synonyms Comment Organism
GDH
-
 Thermus thermophilus
gdh-1
-
 Thermus thermophilus
gdhA_1
-
 Thermus thermophilus
NAD(P)-dependent GDH
-
 Thermus thermophilus
TTC1211
-
 Thermus thermophilus
TTC1212
-
 Thermus thermophilus
TtGDH
-
 Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.2
-
 NADH recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.23
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.27
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, low Glu concentration, pH and temperature not specified in the publication Thermus thermophilus
0.31
-
 NH3 recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
0.66
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
0.72
-
 NADH recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
1.1
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
1.2
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, low Glu concentration, with AMP, pH and temperature not specified in the publication Thermus thermophilus
1.5
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, high Glu concentration, pH and temperature not specified in the publication Thermus thermophilus
2.8
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, pH and temperature not specified in the publication Thermus thermophilus
3.7
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
4.4
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, high Glu concentration, with AMP, pH and temperature not specified in the publication Thermus thermophilus
4.5
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
9.1
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with AMP, pH and temperature not specified in the publication Thermus thermophilus
9.1
-
 NADH recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
12
-
 NAD+ recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
13
-
 L-glutamate recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
15
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
16
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with L-leucine, pH and temperature not specified in the publication Thermus thermophilus
29
-
 NADH recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
37
-
 2-oxoglutarate recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus
44
-
 NH3 recombinant enzyme complex TtGDH-APRTh, with L-leucine and AMP, pH and temperature not specified in the publication Thermus thermophilus

Cofactor

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

General Information

General Information Comment Organism
evolution GDH is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH is believed not to be regulated allosterically because it lacks the antenna helix Thermus thermophilus
malfunction growth analysis of the aprth knockout strain (Tt27DELTAAPRTh) and aprth-overexpressing strain (Tt27NStHisAPRTh) of Thermus thermophilus in minimal medium. The Tt27DELTAAPRTh strain exhibits delayed growth and requires approximately 36 h to reach the early stationary phase, whereas the wild-type strain reaches this phase after 21 h of cultivation. The overexpressing Tt27NStHisAPRTh strain exhibits better growth than even the wild-type strain Thermus thermophilus
additional information glutamate dehydrogenase (GDH) from Thermus thermophilus is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, forms a ternary complex with the enzyme heterodimer. The ternary complex exhibits GDH activity that is activated by leucine, as observed for the GdhA-GdhB binary complex Thermus thermophilus
physiological function GDH catalyzes the synthesis and degradation of glutamate using NAD(P)(H). Bacterial GDH is believed not to be regulated allosterically because it lacks the antenna helix. TtGDH is activated by AMP in a complex with APRTh where APRTh is necessary for the AMP-mediated allosteric activation of TtGDH. L-Leucine is also required for allosteric regulation/activation of the enzyme Thermus thermophilus