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Literature summary for 1.1.1.286 extracted from

  • Shimizu, T.; Yin, L.; Yoshida, A.; Yokooji, Y.; Hachisuka, S.I.; Sato, T.; Tomita, T.; Nishida, H.; Atomi, H.; Kuzuyama, T.; Nishiyama, M.
    Structure and function of an ancestral-type beta-decarboxylating dehydrogenase from Thermococcus kodakarensis (2017), Biochem. J., 474, 105-122.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) 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 (Comment) 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

Protein Variants

Protein Variants Comment 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 Comment 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 Thermococcus kodakarensis
1.5
-
NAD+ 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 Comment Organism Structure
Mn2+ required, binding structure, overview Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 + H+
-
?
isocitrate + NAD+ Thermococcus kodakarensis KRD1
-
2-oxoglutarate + CO2 + NADH + H+
-
?

Organism

Organism UniProt Comment Textmining
Thermococcus kodakarensis Q5JFV8
-
-
Thermococcus kodakarensis KRD1 Q5JFV8
-
-

Purification (Commentary)

Purification (Comment) 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-isopropylmalate + NAD+
-
Thermococcus kodakarensis 2-oxoisocaproate + CO2 + NADH + H+
-
?
3-isopropylmalate + NAD+
-
Thermococcus kodakarensis KRD1 2-oxoisocaproate + CO2 + NADH + H+
-
?
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 + H+
-
?
isocitrate + NAD+
-
Thermococcus kodakarensis KRD1 2-oxoglutarate + CO2 + NADH + H+
-
?
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 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 Thermococcus kodakarensis KRD1 ?
-
?

Subunits

Subunits Comment Organism
More enzyme structure analysis, structure comparisons, overview Thermococcus kodakarensis

Synonyms

Synonyms Comment Organism
HICDH
-
Thermococcus kodakarensis
TK0280
-
Thermococcus kodakarensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
60
-
assay at Thermococcus kodakarensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment 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
-
homoisocitrate pH 8.0, 60°C, recombinant His6-tagged mutant T71V Thermococcus kodakarensis
4.8
-
3-isopropylmalate pH 8.0, 60°C, recombinant His6-tagged wild-type enzyme 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 Comment Organism
8
-
assay at Thermococcus kodakarensis

Cofactor

Cofactor Comment Organism Structure
NAD+
-
Thermococcus kodakarensis

General Information

General Information Comment 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 Comment 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 Thermococcus kodakarensis
3.2
-
NAD+ 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