Information on EC 1.1.1.103 - L-threonine 3-dehydrogenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea

EC NUMBER
COMMENTARY hide
1.1.1.103
-
RECOMMENDED NAME
GeneOntology No.
L-threonine 3-dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-threonine + NAD+ = L-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
aminopropanol phosphate biosynthesis II
-
-
Glycine, serine and threonine metabolism
-
-
L-threonine degradation II
-
-
L-threonine degradation III (to methylglyoxal)
-
-
NIL
-
-
threonine metabolism
-
-
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
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
goat
-
-
Manually annotated by BRENDA team
Japanese quail, fed a standard or threonine-rich diet or fasted for three days
-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
strain KUC-1
Swissprot
Manually annotated by BRENDA team
strain KUC-1
Swissprot
Manually annotated by BRENDA team
strain MDS42, which lacks 14.3% of its chromosome
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-
Manually annotated by BRENDA team
strain MDS42, which lacks 14.3% of its chromosome
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
gene tdh
-
-
Manually annotated by BRENDA team
strain OT3
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Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
-
knockdown of the enzyme and posttranslational downregulation by microRNA-9 inhibits reprogramming efficiency
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetoin + NAD+
butan-2,3-dione + NADH + H+
show the reaction diagram
butane-2,3-diol + NAD+
? + NADH
show the reaction diagram
-
-
-
-
?
D-allothreonine + NAD+
D-2-amino-3-oxobutanoate + NADH
show the reaction diagram
-
-
-
?
D-threonine + NAD+
D-2-amino-3-oxobutyrate + NADH
show the reaction diagram
-
-
-
-
?
DL-2-amino-3-hydroxypentanoate + NAD+
L-2-amino-3-oxopentanoate + NADH
show the reaction diagram
DL-2-amino-3-hydroxyvalerate + NAD+
DL-2-amino-3-oxopentanoate + NADH + H+
show the reaction diagram
DL-2-amino-3-hydroxyvalerate + NAD+
DL-2-amino-3-oxovalerate + NADH
show the reaction diagram
DL-3-hydroxynorvaline + NAD+
3-oxonorvaline + NADH + H+
show the reaction diagram
DL-allothreonine + NAD+
D-2-amino-3-oxobutanoate + NADH
show the reaction diagram
-
-
-
?
DL-threo-3-hydroxynorvaline + NAD+
? + NADH
show the reaction diagram
DL-threo-3-phenylserine + NAD+
? + NADH
show the reaction diagram
DL-threo-beta-phenylserine + NAD+
DL-2-amino-3-phenyl-3-oxopropionate + NADH
show the reaction diagram
DL-threonine hydroxamate + NAD+
DL-2-amino-3-oxobutoxamate + NADH
show the reaction diagram
-
-
-
?
L-allothreonine + NAD+
L-2-amino-3-oxobutanoate + NADH
show the reaction diagram
-
-
-
?
L-serine + NAD+
? + NADH
show the reaction diagram
L-serine + NAD+
L-2-amino-3-oxopropionate + NADH + H+
show the reaction diagram
L-threonine + 3-acetyl-pyridine adenine dinucleotide
L-2-amino-3-oxobutanoate + ?
show the reaction diagram
L-threonine + 3-pyridinealdehyde adenine dinucleotide
L-2-amino-3-oxobutanoate + ?
show the reaction diagram
L-threonine + NAD+
L-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
L-threonine + NAD+
L-2-amino-3-oxobutyrate + NADH + H+
show the reaction diagram
L-threonine + nicotinamide guanine dinucleotide
L-2-amino-3-oxobutanoate + ?
show the reaction diagram
5.1% the rate of NAD+
-
-
?
L-threonine + thionicotinamide-NAD+
L-2-amino-3-oxobutanoate + ?
show the reaction diagram
5.1% the rate of NAD+
-
-
?
L-threonine amide + NAD+
L-2-amino-3-oxobutyramide + NADH
show the reaction diagram
L-threonine methyl ester + NAD+
L-2-amino-3-oxobutanoate methyl ester + NADH
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-threonine + NAD+
L-2-amino-3-oxobutanoate + NADH + H+
show the reaction diagram
L-threonine + NAD+
L-2-amino-3-oxobutyrate + NADH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-acetyl-pyridine adenine dinucleotide
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nicotinamide guanine dinucleotide
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thionicotinamide-NAD+
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
AlCl3
18% activation at 1 mM
BaCl2
13% activation at 1 mM
CoCl2
15% activation at 1 mM
CrCl3
10% activation at 1 mM
CsCl
17% activation at 1 mM
CuSO4
15% activation at 1 mM
MgSO4
12% activation at 1 mM
MnSO4
12% activation at 1 mM
Na2MoO4
13% activation at 1 mM
Ni2+
-
0.1 mM reactivates EDTA-100% inhibited enzyme by 30%
NiCl2
19% activation at 1 mM
PbCl2
18% activation at 1 mM
ZnSO4
9% activation at 1 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
1.26 mM, 41% inhibition after 1 h, 82% inhibition after 2 h, no change in remaining activity after removal of 1,10-phenanthroline
4-chloromercuribenzonic acid
94% inhibition at 10 mM
5,5'-dithiobis-(2-nitrobenzoic acid)
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0.25 mM, 90% inhibition, 67% activity is recovered after incubation with 1 mM, 2-mercaptoethanol or dithieothreitol for 15 min
adenosine-5'-diphosphoribose
aminoacetone
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uncompetitive inhibition vs. NAD+ or L-threonine
Be2+
-
3.2 mM, 20-50% inhibition
calcium pantothenate
slight inhibition
Cd2+
-
0.05 and 1.0 mM, 90% inhibition
Co2+
0.9 mM, 61% inhibiton
CuCl2
1 mM, 100% inhibition
dipicolinic acid
-
40 mM, 99% inhibition after 1 h, complete loss of enzyme-bound Zn2+
FeCl2
32% inhibition at 1 mM after 60 min
FeCl3
73% inhibition at 1 mM after 60 min
HCO3-
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noncompetitive inhibition vs. NAD+ or L-threonine
iodoacetamide
iodoacetate
iodoacetic acid
52% inhibition at 10 mM
Iodosobenzoic acid
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0.3 mM, 17% inhibition
K3[Fe(CN)6]
25% inhibition at 10 mM
L-2-amino-3-oxobutyrate
-
competitive product inhibition by the unstable L-2-amino-3-oxobutyrate only in presence of NADH, which stabilizes
methyl p-nitrobenzenesulfonate
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2 mM, 40% inhibition within 80 min, 65% protection with 250 mM L-threonine, 64% with 250 mM L-threonine methyl ester, 58% with 250 mM L-threonine amide
methylglyoxal
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1.0 mM, 42% inhibition, 2 mM, 63% inhibition, methylglyoxal binds at an allosteric site of the enzyme
methylmethanethiosulfonate
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0.4 mM, 350fold molar excess over enzyme sulfhydryl groups leads to complete inactivation
monoiodoacetate
10 mM, 100% inhibition
N-ethylmaleimide
NAD+
-
competitive inhibition of L-2-amino-3-oxobutanoate reduction
NaN3
88% inhibition at 10 mM
p-chloromercuribenzoate
-
complete inhibition
p-chloromercuribenzoic acid
10 mM, 44% inhibition
p-mercuribenzoate
phenazinemethosulfate
complete inhibition
phenylmethanesulfonyl fluoride
63% inhibition at 10 mM
pyruvate
SnCl2
16% inhibition at 1 mM after 60 min
thionitrobenzoate
-
40fold molar excess, gradual 99% loss of enzyme activity
ZnCl2
1 mM, 72% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
14% activation at 10 mM
DTT
25% activation at 10 mM
additional information
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L-ThrDH is unaffected by EDTA, Li2SO4, MgCl2, MnCl2, CaCl2, NiCl2, CoCl2, BaCl2, HgCl2, CdSO4, CuSO4, ZnCl2, or iodoacetic acid, each at 1 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16
L-allothreonine
-
-
0.0118 - 221
L-threonine
0.0099 - 1
NAD+
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.55 - 551.7
L-threonine
0.009 - 551.7
NAD+
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.37 - 1.42
adenosine-5'-diphosphoribose
23.2 - 50.7
pyruvate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.029
-
partially purified enzyme
0.032
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enzyme activity in mitochondrial extracts prepared from fresh mitochondria
0.055
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enzyme activity in mitochondrial extracts
0.062
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enzyme activity in mitochondrial extracts after freezing the mitochondria for 2 weeks at -20C
0.1
-
C38D mutant, incubated with 1 mM Mn2+ 15 min before assay
0.2
-
C38D mutant
0.6
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C38D mutant, incubated with 0.07 or 5 mM Cd2+ 15 min before assay
1.2
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C38D mutant, incubated with 5 mM Co2+ 15 min before assay
2
-
C38D mutant, incubated with 5 mM Zn2+ and 1 mM Mn2+ 15 min before assay
3.43
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purified recombinant enzyme
8
-
fully demetalized enzyme
8.45
-
-
10.2
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C38D mutant, incubated with 5 mM Zn2+ and 0.07 mM Cd2+ 15 min before assay
11.4
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C38D mutant, incubated with 5 mM Zn2+ 15 min before assay
28.3
-
metal-ion free enzyme
42.2
purified native enzyme, pH 10.0, 30C
65
purified recombinant enzyme, pH 10.0, 30C
91.4
pH 9.0, 10C
156
pH 9.0, 20C
234
L-threonine, pH 9.0, 37C
243
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enzyme activated with 0.07 mM Cd2+
284
pH 9.0, 30C
380
-
-
611
pH 9.0, 50C
685
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enzyme activated with 0.25 mM Mn2+
722
pH 9.0, 40C
18920
-
-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.2 - 8.8
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-
8.4
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C38D mutant enzyme, stimulated with 5 mM Zn2+
8.5 - 8.9
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-
8.6 - 8.7
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30% activity at pH 7.5, 80% activity at pH 9.8
9
-
crude enzyme extracts from mitochondria frozen for 14 days and dialyzed
10.1
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wild-type enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 12
activity range, pH-dependent activity varies in different buffer systems, profile overview
7.4 - 9
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8.5
74% of maximum activity
9.2 - 12
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22% of maximal activity at pH 9.2, 50% at pH 9.5, and 52% at pH 12.0
10.5
31% of maximum activity
additional information
-
pH dependencies of wild-type enzyme and E152D mutant, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
hyperthermophilic enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 30
60 min, more than 90% residual activity
25 - 80
activity range, profile overview
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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extensive threonine utilization
Manually annotated by BRENDA team
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portal drained viscera, PDV
Manually annotated by BRENDA team
additional information
-
no activity in intestine
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Thermococcus kodakarensis (strain ATCC BAA-918 / JCM 12380 / KOD1)
Thermoplasma volcanium (strain ATCC 51530 / DSM 4299 / JCM 9571 / NBRC 15438 / GSS1)
Thermoplasma volcanium (strain ATCC 51530 / DSM 4299 / JCM 9571 / NBRC 15438 / GSS1)
Thermoplasma volcanium (strain ATCC 51530 / DSM 4299 / JCM 9571 / NBRC 15438 / GSS1)
Thermoplasma volcanium (strain ATCC 51530 / DSM 4299 / JCM 9571 / NBRC 15438 / GSS1)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35000
gel filtration, monomeric enzyme
38100
-
sequence analysis
73000
-
gel filtration
74000
-
gel filtration
76000
-
recombinant enzyme, gel filtration
77000
-
gel filtration
78000
-
gel filtration
79400
native enzyme, gel filtration
80000
gel filtration, dimeric enzyme
86000
-
gel filtration
88000
-
density gradient
139000
gel filtration
140000
149000
-
non-denaturing PAGE, wild-type and C38D mutant
154000
-
by gel filtration
155000
-
about, recombinant enzyme, gel filtration
192000
-
recombinant enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
homotetramer
monomer
tetramer
additional information
-
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
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
pro-protein is cleaved to produce a 36000 Da mature enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme, hanging drop vapour diffusion method, 4C, 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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selenomethionine-substituted enzyme, 10 mg/ml protein in 50 mM Tris-HCl buffer, pH 7.5, hanging-drop vapor-diffusion method at 4C, 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
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at room temperature using hanging-drop vapour diffusion method, at 2.4 A resolution. The enzyme is a homotetramer, each monomer consisting of 350 amino acids that form two domains, a catalytic domain and a NAD+-binding domain, which contains an alpha/beta Rossmann fold motif. An extended twelve-stranded beta-sheet is formed by the association of pairs of monomers in the tetramer
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by hanging-drop vapour-diffusion method, to 2.6 A resolution. Crystals grow in the tetragonal space group P43212, with unit-cell parameters a=b=124.5, c=271.1 A. There are four molecules in the asymmetric unit of the crystal
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purified wild-type enzyme or enzyme mutant Y137F in complex with NAD+ and L-3-hydroxynorvaline or pyruvate or L-threonine, sitting drop vapor diffusion method, mixing of 0.001 ml of 40 mg/ml wild-type or 25 mg/ml mutant enzyme, and 1 mM NAD+ mixed with 0.001 ml of 100 mM cacodylate buffer, pH 6.4, 50% v/v 2-methyl-2,4-pentandiol, and 5% w/v PEG 8000, 20C, crystals of ligand-bound wild-type or mutant enzymes by soaking the crystals in reservoir solution containing 0.3 M pyruvate for 2 h, or 0.1ML-threonine for 8 h or 0.1MDL-3-hydroxynorvaline for 7 h, respectively, X-ray diffraction structure determination and analysis at 1.77-2.07 A resolution, molecular replacement
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 11
the purified enzyme is very stable from pH 6.0 to pH 11.0 in the glycine-KOH system, the enzyme is unstable in sodium acetate buffer, pH 4.0-5.0, and Na2CO3-NaHCO3 buffer, pH 10.0-11.0
721272
4.5 - 9.5
-
purified recombinant enzyme, 20 min, 50C, no loss in activity
722747
5.5 - 7.5
-
activity at pH 5.5 is 50% of that at pH 7.5
698526
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 75
quite stable at, rapid loss of activity above 80C
25 - 35
-
85% inactivation after 2 min at 35C, 10% inactivation at 35C in the presence of 80 mM KCl
25 - 45
-
enzyme shows 50% activity at 25C compared with that at 35C
40
half-life 160 min
45
half-life 14 min
50
rapid inactivation
70
-
purified recombinant enzyme, 10 min, 90% activity remaining
80
-
60 min, pH 7.0, over 90% remaining activity, purified recombinant enzyme
85
-
the enzyme shows a half life of 2 h at 85C and 24 min in boiling water
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
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
most stable in Tris-HCl, pH 8, purified preparations in phosphate buffers lose their activity overnight
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 10 mM potassium phosphate, pH 7.0, 30% sucrose, 0.1 mM NAD+, 5 mM L-threonine, several months, no loss of activity
-20C, 14 mM 2-mercaptoethanol, 20% glycerol, at least 1 month, little loss of activity
-
4C, 50 mM Tris-HCl, pH 8.4, 1 mM 2-mercaptoethanol, 2 months, no loss of activity
-
50 mM TrisHCl buffer, pH 8.0
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
acid precipitation, ammonium sulfate, Sephadex G-100, Sephadex G-200
-
ammonium sulfate, DEAE-cellulose, 7fold purification
-
by heat treatment (about 80% pure recombinant TDH protein) and gel filtration
-
by sonication, centrifugation and on a Ni-NTA resin column
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by sonication, centrifugation, heating of the soluble fraction, anion-exchange and hydrophobic interaction chromatography
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by sonication, heating of the cell lysate, anion exchange and hydrophobic interaction chromatography
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chromatography on Q-Sepharose and Reactive Green 19-agarose
DEAE Sepharose FF, Affi-Gel blue, Sephacryl S-200, Matrix Gel red A, Matrix gel Green A
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DEAE-cellulose, ammonium sulfate, DEAE-cellulose, Blue dextran-Sepharose, Sephadex G-200, amylamine sepharose, Sephadex G-200
-
DEAE-cellulose, hydrophobic interaction chromatography, Affi-Gel Blue, Matrex Red A
-
hydroxyapatite
-
MnCl2, ammonium sulfate, calcium phosphate gel, protamine sulfate, DEAE-cellulose, 7fold purification
-
protamine sulfate, Sephadex G-200, DEAE-cellulose
-
recombinant enzyme 48fold from Escherichia coli to homogeneity by heat treatment and anion exchange chromatography
-
recombinant enzyme from Escherichia coli
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by heat treatment at 85C for 30 min, centrifugation, ion exchange chromatography, and gel filtration
-
recombinant His-tagged enzyme 4.2fold from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, native enzyme 75.4fold by anion exchange and hydrophobic interaction chromatography, dialysis, hydroxyapatite chromatography, followed by gel filtration
recombinant His-tagged enzyme from Escherichia coli strain TOP10 by nickel affinity chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by heat treatment at 85C for 30 min, anion exchange and hydrophobic interaction chromatography
-
treatment of cell extract at 60C, DEAE-Sephadex, Mn2+ activation, Blue 2-Sepharose CL-6B, C38D mutant was purified without Mn2+ activation
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned as a 3562-base pair EcoRI fragment
-
DNA and amino acid seuence determination and analysis, sequence comparisons, phylogenetic tree, expression of His-tagged enzyme in Escherichia coli strains JM109 and BL21(DE3)
gene tdh, functional expression in Escherichia coli
-
into pET-30a digested with KpnIEcoRI to construct pET-ste11 and expressed in Escherichia coli BL21
-
ligated into the pTZ57R/T cloning vector. The recombinant pTZ-tdh plasmid digested with NcoI and EcoRI and the TDH gene ligated with pET-8c, giving the recombinant pET-tdh plasmid, used to transform Escherichia coli DH5alpha competent cells. Recombinant and purified pET-tdh plasmid expressed in Escherichia coli BL21 (DE3)
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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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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)
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orf PH0655, overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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overexpression in Escherichia coli
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plasmid pTZ-tdh transformed in Escherichia coli DH5alpha competent cells. NcoI-EcoRI restriction fragment inserted into the pET-8c expression vector at the corresponding sites. The resulting plasmid pET-tdh expressed in Escherichia coli strain BL21(DE3)CodonPlus-RIL
-
recombinant pET-tdh plasmid expressed in Escherichia coli BL21 (DE3)
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C38D
-
site-directed mutagenesis
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
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
Show AA Sequence (3103 entries)
Please use the Sequence Search for a certain query.