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)
EC Number
Commentary
Organism
1.1.1.286
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)
EC Number
Crystallization
Organism
1.1.1.286
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
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.286
I82N
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
L81P
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
L83R
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
S80A
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
T71V
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.286
additional information
-
additional information
Michaelis-Menten kinetics
Thermococcus kodakarensis
1.1.1.286
0.0073
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.014
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.036
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.037
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.042
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.05
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.16
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
0.25
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.3
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.31
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.32
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.46
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
0.5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.58
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
0.6
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.62
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.64
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.68
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.71
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.76
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.92
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.94
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.3
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
1.1.1.286
2.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.286
Mn2+
required, binding structure, overview
Thermococcus kodakarensis
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.286
homoisocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
1.1.1.286
homoisocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
1.1.1.286
isocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoglutarate + CO2 + NADH
-
-
?
1.1.1.286
isocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoglutarate + CO2 + NADH
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.286
Thermococcus kodakarensis
Q5JFV8
-
-
1.1.1.286
Thermococcus kodakarensis KRD1
Q5JFV8
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.286
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)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.286
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
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
?
-
-
-
-
1.1.1.286
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
EC Number
Subunits
Commentary
Organism
1.1.1.286
More
enzyme structure analysis, structure comparisons, overview
Thermococcus kodakarensis
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.286
60
-
assay at
Thermococcus kodakarensis
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.22
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.71
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
1.1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
1.5
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
2.3
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
4.8
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
4.8
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
6.6
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
6.9
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
9.4
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
13
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
43
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
45
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.286
8
-
assay at
Thermococcus kodakarensis
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.286
NAD+
-
Thermococcus kodakarensis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.286
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)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.286
NAD+
-
Thermococcus kodakarensis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
1.1.1.286
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)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.286
I82N
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
L81P
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
L83R
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
S80A
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
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)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.286
additional information
-
additional information
Michaelis-Menten kinetics
Thermococcus kodakarensis
1.1.1.286
0.0073
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.014
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.036
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.037
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.042
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.05
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.16
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
0.25
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.3
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
0.31
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
0.32
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.46
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
0.5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.58
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
0.6
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.62
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.64
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.68
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.71
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.76
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.92
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
0.94
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
1
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.3
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
1.1.1.286
2.2
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.286
Mn2+
required, binding structure, overview
Thermococcus kodakarensis
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.286
homoisocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
1.1.1.286
homoisocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoadipate + CO2 + NADH + H+
-
-
?
1.1.1.286
isocitrate + NAD+
Thermococcus kodakarensis
-
2-oxoglutarate + CO2 + NADH
-
-
?
1.1.1.286
isocitrate + NAD+
Thermococcus kodakarensis KRD1
-
2-oxoglutarate + CO2 + NADH
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.286
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)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.286
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
3-isopropylmalate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoisocaproate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoadipate + CO2 + NADH + H+
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
740021
Thermococcus kodakarensis
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
740021
Thermococcus kodakarensis KRD1
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
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
?
-
-
-
-
1.1.1.286
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)
EC Number
Subunits
Commentary
Organism
1.1.1.286
More
enzyme structure analysis, structure comparisons, overview
Thermococcus kodakarensis
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.286
60
-
assay at
Thermococcus kodakarensis
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.22
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.71
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
1.1
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
1.5
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
2.3
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
4.8
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
4.8
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
5
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
6.6
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
6.9
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
9.4
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
13
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
17
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
43
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
45
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.286
8
-
assay at
Thermococcus kodakarensis
General Information
EC Number
General Information
Commentary
Organism
1.1.1.286
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
1.1.1.286
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
1.1.1.286
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)
EC Number
General Information
Commentary
Organism
1.1.1.286
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
1.1.1.286
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
1.1.1.286
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]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.44
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.45
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
0.56
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.88
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.92
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.93
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.1.1.286
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.1.1.286
1.9
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
2.2
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
2.4
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
5.3
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
7.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
9.4
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
11
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
11
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
1.1.1.286
13
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
15
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
41
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
46
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
50
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
1.1.1.286
70
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
98
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
190
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
250
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
270
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
310
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
360
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
470
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
2300
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.286
0.44
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
0.45
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
0.56
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.88
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with isocitrate
Thermococcus kodakarensis
1.1.1.286
0.92
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
0.93
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with isocitrate
Thermococcus kodakarensis
1.1.1.286
1.2
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
1.8
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with isocitrate
Thermococcus kodakarensis
1.1.1.286
1.9
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
2.2
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
2.4
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
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
1.1.1.286
5.3
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
7.5
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant S80A, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
9.4
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant T71V, with 3-isopropylmalate
Thermococcus kodakarensis
1.1.1.286
11
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
11
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with isocitrate
Thermococcus kodakarensis
1.1.1.286
13
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant T71V
Thermococcus kodakarensis
1.1.1.286
15
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
41
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L81P, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
46
-
3-isopropylmalate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
50
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with isocitrate
Thermococcus kodakarensis
1.1.1.286
70
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant I82N, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
98
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant I82N
Thermococcus kodakarensis
1.1.1.286
190
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant S80A
Thermococcus kodakarensis
1.1.1.286
250
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L83R
Thermococcus kodakarensis
1.1.1.286
270
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged mutant L83R, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
310
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged mutant L81P
Thermococcus kodakarensis
1.1.1.286
360
-
isocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis
1.1.1.286
470
-
NAD+
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme, with homoisocitrate
Thermococcus kodakarensis
1.1.1.286
2300
-
homoisocitrate
pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme
Thermococcus kodakarensis