Cloned (Comment) | Organism |
---|---|
orf PH0655, NA and amino acid sequence determination, analysis, and comparison, expression of selenomethionine-labeled wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Pyrococcus horikoshii |
Crystallization (Comment) | Organism |
---|---|
selenomethionine-substituted enzyme, 10 mg/ml protein in 50 mM Tris-HCl buffer, pH 7.5, hanging-drop vapor-diffusion method at 4°C, mixing of 0.0015 ml of each, protein and reservoir solution, the latter containing 0.2 M sodium chloride, 0.1 M HEPES buffer, pH 7.5, and 40% v/v PEG 400, five days, X-ray diffraction structure determination and analysis, single wavelength anomalous diffraction method | Pyrococcus horikoshii |
Protein Variants | Comment | Organism |
---|---|---|
E199A | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Pyrococcus horikoshii |
R204A | site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme | Pyrococcus horikoshii |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0099 | - |
NAD+ | pH 7.5, 65°C, recombinant wild-type enzyme | Pyrococcus horikoshii | |
0.0118 | - |
L-threonine | pH 7.5, 65°C, recombinant wild-type enzyme | Pyrococcus horikoshii | |
0.291 | - |
NAD+ | pH 7.5, 65°C, recombinant mutant R204A | Pyrococcus horikoshii | |
0.304 | - |
NAD+ | pH 7.5, 65°C, recombinant mutant E199A | Pyrococcus horikoshii | |
1.51 | - |
L-threonine | pH 7.5, 65°C, recombinant mutant E199A | Pyrococcus horikoshii | |
2.5 | - |
L-threonine | pH 7.5, 65°C, recombinant mutant R204A | Pyrococcus horikoshii |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | one molecule of TDH contains one zinc ion playing a structural role, the metal ion is ligated by foru Cys residues, coenzyme-binding domain shows a larger interdomain cleft compared to other ADHs, binding structure, overview | Pyrococcus horikoshii |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
35000 | - |
4 * 35000, recombinant enzyme, SDS-PAGE | Pyrococcus horikoshii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-threonine + NAD+ | Pyrococcus horikoshii | - |
(2S)-2-amino-3-oxobutanoate + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pyrococcus horikoshii | O58389 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-threonine + NAD+ | - |
Pyrococcus horikoshii | (2S)-2-amino-3-oxobutanoate + NADH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
More | crystal structure analysis, each subunit is composed of two domains: an NAD(H)-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein | Pyrococcus horikoshii |
tetramer | 4 * 35000, recombinant enzyme, SDS-PAGE | Pyrococcus horikoshii |
Synonyms | Comment | Organism |
---|---|---|
L-threonine dehydrogenase | - |
Pyrococcus horikoshii |
More | the enzyme belongs to the medium-chain NAD(H)-dependent alcohol dehydrogenases | Pyrococcus horikoshii |
TDH | - |
Pyrococcus horikoshii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | - |
assay at | Pyrococcus horikoshii |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.55 | - |
NAD+ | pH 7.5, 65°C, recombinant mutant E199A | Pyrococcus horikoshii | |
0.55 | - |
L-threonine | pH 7.5, 65°C, recombinant mutant E199A | Pyrococcus horikoshii | |
0.667 | - |
NAD+ | pH 7.5, 65°C, recombinant mutant R204A | Pyrococcus horikoshii | |
0.667 | - |
L-threonine | pH 7.5, 65°C, recombinant mutant R204A | Pyrococcus horikoshii | |
11 | - |
NAD+ | pH 7.5, 65°C, recombinant wild-type enzyme | Pyrococcus horikoshii | |
11 | - |
L-threonine | pH 7.5, 65°C, recombinant wild-type enzyme | Pyrococcus horikoshii |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Pyrococcus horikoshii |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Pyrococcus horikoshii | |
NADH | - |
Pyrococcus horikoshii |