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 (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 TrisHCl, 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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | required, binding structure, overview | Thermococcus kodakarensis |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Thermococcus kodakarensis | Q5JFV8 | - |
- |
Thermococcus kodakarensis KRD1 | Q5JFV8 | - |
- |
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 | 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 | Comment | Organism |
---|---|---|
More | enzyme structure analysis, structure comparisons, overview | Thermococcus kodakarensis |
Synonyms | Comment | Organism |
---|---|---|
HICDH | - |
Thermococcus kodakarensis |
TK0280 | - |
Thermococcus kodakarensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
assay at | Thermococcus kodakarensis |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Thermococcus kodakarensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Thermococcus kodakarensis |
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 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 |