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Literature summary 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)

EC Number Cloned (Comment) 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 (Comment) 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

Protein Variants

EC Number Protein Variants Comment 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 Comment 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 Thermococcus kodakarensis
1.1.1.286 1.5
-
 NAD+ 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 Comment Organism Structure
1.1.1.286 Mn2+ required, binding structure, overview Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.286 Thermococcus kodakarensis Q5JFV8
-
-
1.1.1.286 Thermococcus kodakarensis KRD1 Q5JFV8
-
-

Purification (Commentary)

EC Number Purification (Comment) 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.286 3-isopropylmalate + NAD+
-
 Thermococcus kodakarensis 2-oxoisocaproate + CO2 + NADH + H+
-
 ?
1.1.1.286 3-isopropylmalate + NAD+
-
 Thermococcus kodakarensis KRD1 2-oxoisocaproate + CO2 + NADH + H+
-
 ?
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 + H+
-
 ?
1.1.1.286 isocitrate + NAD+
-
 Thermococcus kodakarensis KRD1 2-oxoglutarate + CO2 + NADH + H+
-
 ?
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 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 Thermococcus kodakarensis KRD1 ?
-
 ?

Subunits

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

Synonyms

EC Number Synonyms Comment Organism
1.1.1.286 HICDH
-
 Thermococcus kodakarensis
1.1.1.286 TK0280
-
 Thermococcus kodakarensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment 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 Comment 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
-
 homoisocitrate pH 8.0, 60°C, recombinant His6-tagged mutant T71V 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 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 Comment Organism
1.1.1.286 8
-
 assay at Thermococcus kodakarensis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.286 NAD+
-
 Thermococcus kodakarensis

General Information

EC Number General Information Comment 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 Comment 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 Thermococcus kodakarensis
1.1.1.286 3.2
-
 NAD+ 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