This enzyme acts in concert with EC 2.3.1.29, glycine C-acetyltransferase, in the degradation of threonine to glycine. This threonine-degradation pathway is common to prokaryotic and eukaryotic cells and the two enzymes involved form a complex . In aqueous solution, the product L-2-amino-3-oxobutanoate can spontaneously decarboxylate to form aminoacetone.
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The taxonomic range for the selected organisms is: Pyrococcus horikoshii The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
catalytic mechanism, the carboxyl group of Glu152 is important for expressing the catalytic activity, the proton relay system works as a catalytic mechanism of TDH
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SYSTEMATIC NAME
IUBMB Comments
L-threonine:NAD+ oxidoreductase
This enzyme acts in concert with EC 2.3.1.29, glycine C-acetyltransferase, in the degradation of threonine to glycine. This threonine-degradation pathway is common to prokaryotic and eukaryotic cells and the two enzymes involved form a complex [2]. In aqueous solution, the product L-2-amino-3-oxobutanoate can spontaneously decarboxylate to form aminoacetone.
1.22 mol of Zn2+ per mol of enzyme subunit, the catalytic zinc atom at the active center of TDH is coordinated by the highly conserved four residues Cys42, His67, Glu68 and Glu152 with low affinity
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
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
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
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
purified recombinant enzyme, hanging drop vapour diffusion method, 4°C, 10 mg/ml protein in 50 mM Tris-HCl, pH 7.5, 0.0015 ml of protein and reservoir solution are mixed, the reservoir solution contains 0.2 M NaCl, 0.1 M HEPES, pH 7.5, and 40% v/v PEG 400, 5 days, X-ray diffraction structure determination and preliminary analysis at 2.2 A resolution
site-directed mutagenesis, the E152D mutant shows 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD+ compared to wild-type TDH, Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD+ complex
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by heat treatment at 85°C for 30 min, anion exchange and hydrophobic interaction chromatography
recombinant enzyme from Escherichia coli strain BL21(DE3) by heat treatment at 85°C for 30 min, centrifugation, ion exchange chromatography, and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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)