BRENDA - Enzyme Database
show all sequences of 1.1.1.286

Structure and function of an ancestral-type beta-decarboxylating dehydrogenase from Thermococcus kodakarensis

Shimizu, T.; Yin, L.; Yoshida, A.; Yokooji, Y.; Hachisuka, S.I.; Sato, T.; Tomita, T.; Nishida, H.; Atomi, H.; Kuzuyama, T.; Nishiyama, M.; Biochem. J. 474, 105-122 (2017)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene TK0280, phylogenetic analysis and tree, cloning in Escherichia coli strain DH5alpha, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-CodonPlus (DE3)-RIL
Thermococcus kodakarensis
Crystallization (Commentary)
Crystallization
Organism
purified recombinant His6-tagged enzyme in apoform or in complex with substrates isocitrate, homoisocitrate, and 3-isopropylmalate, hanging drop vapour diffusion method, for the apoenzyme: mixing of 0.001 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl, with 0.001 ml of reservoir solution containing 100 mM imidazole-HCl, pH 6.5, and 2.3 M NaCl, equilibration against 1 m of reservoir solution, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM homoisocitrate, with 0.0015 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 10% v/v 2-propanol, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 5 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM isocitrate, with 0.0015 ml of reservoir solution containing 100 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 30% v/v 2-methyl-2,4-pentanediol, at 20°C, and for the ternary complex with 3-isopropylmalate and Mn2+: mixing of 0.001 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM 3-isopropylmalate, with 0.001 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 36% v/v 2-methyl-2,4-pentanediol, at 20°C, X-ray diffraction structure determination and analysis at 1.7 A resolution for the apoenzyme, and at 2.50-2.64 A resolution for the complexed enzyme
Thermococcus kodakarensis
Engineering
Amino acid exchange
Commentary
Organism
I82N
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
L81P
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
L83R
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
additional information
generation of a gene-disruption TK0280 deletion mutan strain, functional complementation by expression of the gene icdh from Thermus thermophilus strain Hb27, but not by genes hicdh and leuB from this organism
Thermococcus kodakarensis
S80A
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
T71V
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Thermococcus kodakarensis
0.0073
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.014
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.036
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
0.037
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.042
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.05
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
0.16
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
0.17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
0.25
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
0.3
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.31
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.32
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
0.46
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
0.5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.58
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
0.6
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
0.62
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
0.64
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
0.68
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.71
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.76
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
0.92
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
0.94
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.3
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with homoisocitrate; pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with 3-isopropylmalate
Thermococcus kodakarensis
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
2.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Mn2+
required, binding structure, overview
Thermococcus kodakarensis
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
homoisocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
homoisocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
isocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoglutarate + CO2 + NADH
-
-
?
isocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoglutarate + CO2 + NADH
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Thermococcus kodakarensis
Q5JFV8
-
-
Thermococcus kodakarensis KRD1
Q5JFV8
-
-
Purification (Commentary)
Commentary
Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus (DE3)-RIL by nickel affinity chromatography and gel filtration
Thermococcus kodakarensis
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
isocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoglutarate + CO2 + NADH
-
-
-
?
isocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoglutarate + CO2 + NADH
-
-
-
?
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280
740021
Thermococcus kodakarensis
?
-
-
-
-
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280
740021
Thermococcus kodakarensis KRD1
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
More
enzyme structure analysis, structure comparisons, overview
Thermococcus kodakarensis
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
60
-
assay at
Thermococcus kodakarensis
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.22
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.71
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.5
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
2.3
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
4.8
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
4.8
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
6.6
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
6.9
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
9.4
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
13
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
43
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
45
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Thermococcus kodakarensis
Cofactor
Cofactor
Commentary
Organism
Structure
NAD+
-
Thermococcus kodakarensis
Cloned(Commentary) (protein specific)
Commentary
Organism
gene TK0280, phylogenetic analysis and tree, cloning in Escherichia coli strain DH5alpha, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-CodonPlus (DE3)-RIL
Thermococcus kodakarensis
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NAD+
-
Thermococcus kodakarensis
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified recombinant His6-tagged enzyme in apoform or in complex with substrates isocitrate, homoisocitrate, and 3-isopropylmalate, hanging drop vapour diffusion method, for the apoenzyme: mixing of 0.001 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 150 mM NaCl, with 0.001 ml of reservoir solution containing 100 mM imidazole-HCl, pH 6.5, and 2.3 M NaCl, equilibration against 1 m of reservoir solution, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 10 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM homoisocitrate, with 0.0015 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 10% v/v 2-propanol, at 20°C, for the ternary complex with homoisocitrate and Mn2+: mixing of 0.0015 ml of 5 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM isocitrate, with 0.0015 ml of reservoir solution containing 100 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 30% v/v 2-methyl-2,4-pentanediol, at 20°C, and for the ternary complex with 3-isopropylmalate and Mn2+: mixing of 0.001 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM MnSO4, and 5 mM 3-isopropylmalate, with 0.001 ml of reservoir solution containing 100 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, and 36% v/v 2-methyl-2,4-pentanediol, at 20°C, X-ray diffraction structure determination and analysis at 1.7 A resolution for the apoenzyme, and at 2.50-2.64 A resolution for the complexed enzyme
Thermococcus kodakarensis
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
I82N
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
L81P
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
L83R
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
additional information
generation of a gene-disruption TK0280 deletion mutan strain, functional complementation by expression of the gene icdh from Thermus thermophilus strain Hb27, but not by genes hicdh and leuB from this organism
Thermococcus kodakarensis
S80A
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
T71V
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
Michaelis-Menten kinetics
Thermococcus kodakarensis
0.0073
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.014
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.036
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
0.037
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.042
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.05
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
0.16
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
0.17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
0.25
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
0.3
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
0.31
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
0.32
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
0.46
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
0.5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.58
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
0.6
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
0.62
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
0.64
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
0.68
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.71
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.76
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
0.92
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
0.94
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.3
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with homoisocitrate; pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with 3-isopropylmalate
Thermococcus kodakarensis
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
2.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Mn2+
required, binding structure, overview
Thermococcus kodakarensis
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
homoisocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
homoisocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
isocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoglutarate + CO2 + NADH
-
-
?
isocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoglutarate + CO2 + NADH
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus (DE3)-RIL by nickel affinity chromatography and gel filtration
Thermococcus kodakarensis
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
isocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoglutarate + CO2 + NADH
-
-
-
?
isocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoglutarate + CO2 + NADH
-
-
-
?
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280
740021
Thermococcus kodakarensis
?
-
-
-
-
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280
740021
Thermococcus kodakarensis KRD1
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
More
enzyme structure analysis, structure comparisons, overview
Thermococcus kodakarensis
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
60
-
assay at
Thermococcus kodakarensis
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.22
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.71
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.5
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
2.3
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
4.8
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
4.8
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
6.6
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
6.9
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
9.4
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
13
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
43
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
45
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
8
-
assay at
Thermococcus kodakarensis
General Information
General Information
Commentary
Organism
evolution
TK0280 from Thermococcus kodakarensis is an ancestral-type beta-decarboxylating dehydrogenase. beta-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity, phylogenetic analysis, overview
Thermococcus kodakarensis
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280. Structural basis of substrate promiscuity, conformational changes upon binding of substrates and active site structure, overview
Thermococcus kodakarensis
physiological function
recombinant enzyme TK0280 exhibits dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in Thermococcus kodakarensis, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate
Thermococcus kodakarensis
General Information (protein specific)
General Information
Commentary
Organism
evolution
TK0280 from Thermococcus kodakarensis is an ancestral-type beta-decarboxylating dehydrogenase. beta-Decarboxylating dehydrogenases, which are involved in central metabolism, are considered to have diverged from a common ancestor with broad substrate specificity, phylogenetic analysis, overview
Thermococcus kodakarensis
additional information
residues Thr71 and Ser80 play important roles in the recognition of homoisocitrate and isocitrate while the hydrophobic region consisting of Ile82 and Leu83 is responsible for the recognition of 3-isopropylmalate. Importance of a water-mediated hydrogen bond network for the stabilization of the beta3-alpha4 loop, including the Thr71 residue, with respect to the promiscuity of the substrate specificity of TK0280. Structural basis of substrate promiscuity, conformational changes upon binding of substrates and active site structure, overview
Thermococcus kodakarensis
physiological function
recombinant enzyme TK0280 exhibits dehydrogenase activities toward homoisocitrate, isocitrate, and 3-isopropylmalate, which correspond to key reactions involved in the lysine biosynthetic pathway, tricarboxylic acid cycle, and leucine biosynthetic pathway, respectively. TK0280 functions as both an isocitrate dehydrogenase and homoisocitrate dehydrogenase in Thermococcus kodakarensis, but not as a 3-isopropylmalate dehydrogenase, most probably reflecting its low catalytic efficiency toward 3-isopropylmalate
Thermococcus kodakarensis
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.44
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.45
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
0.56
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
0.88
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
0.92
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.93
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.9
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
2.2
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
2.4
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
3.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with homoisocitrate; pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with 3-isopropylmalate
Thermococcus kodakarensis
5.3
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
7.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
9.4
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
11
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
11
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
13
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
15
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
41
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
46
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
50
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
70
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
98
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
190
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
250
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
270
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
310
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
360
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
470
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
2300
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.44
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
0.45
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
0.56
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
0.88
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
0.92
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
0.93
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.9
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
2.2
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
2.4
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
3.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with homoisocitrate; pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with 3-isopropylmalate
Thermococcus kodakarensis
5.3
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
7.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
9.4
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
11
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
11
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
13
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
15
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
41
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
46
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
50
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
70
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
98
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
190
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
250
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
270
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
310
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
360
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
470
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
2300
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
Other publictions for EC 1.1.1.286
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
740021
Shimizu
Structure and function of an a ...
Thermococcus kodakarensis, Thermococcus kodakarensis KRD1
Biochem. J.
474
105-122
2017
-
-
1
1
6
-
-
32
-
1
-
4
-
5
-
-
1
-
-
-
-
-
8
1
1
-
-
16
1
-
-
1
-
-
-
-
-
1
1
1
6
-
-
-
-
32
-
1
-
4
-
-
-
1
-
-
-
-
8
1
1
-
-
16
1
-
-
-
-
3
3
-
31
31
738195
Takahashi
Characterization of two ?-deca ...
Sulfolobus acidocaldarius, Sulfolobus acidocaldarius MW001
Extremophiles
20
843-853
2016
-
-
1
-
-
-
-
2
-
-
-
6
-
5
-
-
1
-
-
-
-
-
6
1
1
-
-
2
1
-
-
2
-
-
-
-
-
1
2
-
-
-
-
-
-
2
-
-
-
6
-
-
-
1
-
-
-
-
6
1
1
-
-
2
1
-
-
-
-
1
1
-
2
2
740000
Takahashi
Determinants of dual substrate ...
Thermus thermophilus, Thermus thermophilus DSM 7039
Biochem. Biophys. Res. Commun.
478
1688-1693
2016
-
-
1
1
-
-
1
2
-
1
-
6
-
2
-
-
1
-
-
-
-
-
8
1
1
-
-
2
1
-
-
2
-
-
-
-
-
1
2
1
-
-
-
1
-
2
-
1
-
6
-
-
-
1
-
-
-
-
8
1
1
-
-
2
1
-
-
-
-
3
3
-
2
2
685232
Lin
Chemical mechanism of homoisoc ...
Saccharomyces cerevisiae
Biochemistry
47
4169-4180
2008
-
-
-
-
-
-
3
1
-
-
-
1
-
1
-
-
-
2
-
-
-
-
3
-
1
-
-
-
1
1
-
1
1
-
-
-
-
-
1
-
-
-
-
3
1
1
-
-
-
1
-
-
-
-
-
-
-
-
3
-
1
-
-
-
1
1
-
-
-
-
-
-
-
-
696220
Lin
Potassium is an activator of h ...
Saccharomyces cerevisiae
Biochemistry
47
10809-10815
2008
-
-
-
-
-
-
4
-
-
4
-
1
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
1
3
-
-
-
-
-
1
-
-
-
-
4
3
-
-
4
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
660907
Miyazaki
Bifunctional isocitrate-homois ...
Pyrococcus horikoshii
Biochem. Biophys. Res. Commun.
331
341-346
2005
-
-
1
-
-
-
-
3
-
-
-
-
-
3
-
-
1
-
-
-
-
-
4
-
-
-
-
3
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
1
-
-
-
-
4
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
660918
Miyazaki
Identification of a novel trif ...
Deinococcus radiodurans
Biochem. Biophys. Res. Commun.
336
596-602
2005
-
-
-
-
4
-
3
8
-
3
3
-
-
1
-
-
-
-
-
-
-
-
5
1
-
-
-
8
-
-
-
1
-
-
-
-
-
-
1
-
4
-
-
3
-
8
-
3
3
-
-
-
-
-
-
-
-
-
5
1
-
-
-
8
-
-
-
-
-
-
-
-
-
-
656127
Miyazaki
Characterization of homoisocit ...
Thermus thermophilus, Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039
J. Biol. Chem.
278
1864-1871
2003
-
-
1
-
1
-
-
2
-
-
-
-
-
2
-
-
-
-
-
-
-
-
6
1
-
-
1
2
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
6
1
-
-
1
2
-
-
-
-
-
-
-
-
-
-