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Information on EC 1.1.1.103 - L-threonine 3-dehydrogenase and Organism(s) Pyrococcus horikoshii and UniProt Accession O58389

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IUBMB Comments
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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This record set is specific for:
Pyrococcus horikoshii
UNIPROT: O58389
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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
Synonyms
threonine dehydrogenase, l-threonine dehydrogenase, l-threonine 3-dehydrogenase, l-thrdh, thrdh, orf382, threonine 3-dehydrogenase, thr dehydrogenase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
L-threonine dehydrogenase
-
L-threonine dehydrogenase
threonine 3-dehydrogenase
-
-
-
-
threonine dehydrogenase
-
-
-
-
additional information
the enzyme belongs to the medium-chain NAD(H)-dependent alcohol dehydrogenases
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-threonine + NAD+ = L-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
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
L-threonine + NAD+ = L-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
reaction mechanism, random bi bi kinetic mechanism
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
-
PATHWAY SOURCE
PATHWAYS
-
-, -, -
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.
CAS REGISTRY NUMBER
COMMENTARY hide
9067-99-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-threonine + NAD+
(2S)-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
DL-threo-3-phenylserine + NAD+
? + NADH
show the reaction diagram
-
53% of the activity with L-threonine
-
-
?
L-serine + NAD+
? + NADH
show the reaction diagram
-
21% of the activity with L-threonine
-
-
?
L-threonine + NAD+
(2S)-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-threonine + NAD+
(2S)-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
L-threonine + NAD+
(2S)-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
-
-
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
0.54 mol Ca2+ per mol of enzyme subunit
Co2+
-
can replace Zn2+ with 35% of the activity with Zn2+
Cu2+
-
0.29 mol Ca2+ per mol of enzyme subunit
Mg2+
-
0.2 mol Ca2+ per mol of enzyme subunit
Mn2+
-
can replace Zn2+ with 77% of the activity with Zn2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
-
complete inhibition at 50 mM
L-2-amino-3-oxobutyrate
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competitive product inhibition by the unstable L-2-amino-3-oxobutyrate only in presence of NADH, which stabilizes
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0118 - 2.5
L-threonine
0.0099 - 0.607
NAD+
0.013
L-threonine
-
pH 7.5, 65°C
0.01
NAD+
-
pH 7.5, 65°C
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.55 - 11
L-threonine
0.009 - 11
NAD+
7.2 - 551.7
L-threonine
7.2 - 551.7
NAD+
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.43
-
purified recombinant enzyme
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.2 - 12
-
22% of maximal activity at pH 9.2, 50% at pH 9.5, and 52% at pH 12.0
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
hyperthermophilic enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
4 * 35000, recombinant enzyme, SDS-PAGE
38000
x * 38000, recombinant wild-type and mutant enzymes, SDS-PAGE
192000
-
recombinant enzyme, gel filtration
37700
-
x * 37700, recombinant enzyme, SDS-PAGE
37742
-
2 * 36000-38000, recombinant enzyme, SDS-PAGE, 2 * 37742, sequence calculation
41200
-
4 * 41200, recombinant enzyme, SDS-PAGE, 4 * 41815, sequence calculation
41815
-
4 * 41200, recombinant enzyme, SDS-PAGE, 4 * 41815, sequence calculation
76000
-
recombinant enzyme, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 38000, recombinant wild-type and mutant enzymes, SDS-PAGE
tetramer
4 * 35000, recombinant enzyme, SDS-PAGE
?
-
x * 37700, recombinant enzyme, SDS-PAGE
dimer
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2 * 36000-38000, recombinant enzyme, SDS-PAGE, 2 * 37742, sequence calculation
tetramer
-
4 * 41200, recombinant enzyme, SDS-PAGE, 4 * 41815, sequence calculation
additional information
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
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E152A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E152C
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E152D
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
E152K
site-directed mutagenesis, inactive mutant
E152Q
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E152S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E152T
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E199A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R204A
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
100
-
30 min, pH 7.0, over 90% remaining activity, purified recombinant enzyme
105
-
higher loss of activity above, purified recombinant enzyme
108
-
2-step thermal inactivation
120
-
20 min, inactivation, purified recombinant enzyme
80
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60 min, pH 7.0, over 90% remaining activity, purified recombinant enzyme
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
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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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recombinant His-tagged enzyme from Escherichia coli strain TOP10 by nickel affinity chromatography
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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)
orf PH0655, overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
orf PH0655, amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain TOP10
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orf PH0655, expression in Escherichia coli
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orf PH0655, gene TDH, overexpression in Escherichia coli strain BL21(DE3)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Higashi, N.; Matsuura, T.; Nakagawa, A.; Ishikawa, K.
Crystallization and preliminary X-ray analysis of hyperthermophilic L-threonine dehydrogenase from the archaeon Pyrococcus horikoshii
Acta crystallogr. Sect. F
61
432-434
2005
Pyrococcus horikoshii
Manually annotated by BRENDA team
Shimizu, Y.; Sakuraba, H.; Kawakami, R.; Goda, S.; Kawarabayasi, Y.; Ohshima, T.
L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization
Extremophiles
9
317-324
2005
Pyrococcus horikoshii, Pyrococcus horikoshii OT-3
Manually annotated by BRENDA team
Higashi, N.; Fukada, H.; Ishikawa, K.
Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii
J. Biosci. Bioeng.
99
175-180
2005
Pyrococcus horikoshii
Manually annotated by BRENDA team
Higashi, N.; Tanimoto, K.; Nishioka, M.; Ishikawa, K.; Taya, M.
Investigating a catalytic mechanism of hyperthermophilic L-threonine dehydrogenase from Pyrococcus horikoshii
J. Biochem.
144
77-85
2008
Pyrococcus horikoshii (O58389), Pyrococcus horikoshii
Manually annotated by BRENDA team
Ishikawa, K.; Higashi, N.; Nakamura, T.; Matsuura, T.; Nakagawa, A.
The first crystal structure of L-threonine dehydrogenase
J. Mol. Biol.
366
857-867
2007
Pyrococcus horikoshii (O58389), Pyrococcus horikoshii
Manually annotated by BRENDA team