Information on EC 1.4.1.9 - leucine dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.4.1.9
-
RECOMMENDED NAME
GeneOntology No.
leucine dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-leucine + H2O + NAD+ = 4-methyl-2-oxopentanoate + NH3 + NADH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
leucine metabolism
-
-
Valine, leucine and isoleucine degradation
-
-
Valine, leucine and isoleucine biosynthesis
-
-
Metabolic pathways
-
-
Biosynthesis of secondary metabolites
-
-
Biosynthesis of antibiotics
-
-
SYSTEMATIC NAME
IUBMB Comments
L-leucine:NAD+ oxidoreductase (deaminating)
Also acts on isoleucine, valine, norvaline and norleucine.
CAS REGISTRY NUMBER
COMMENTARY hide
9082-71-7
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
Bacillus niger
-
-
-
Manually annotated by BRENDA team
168M
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain ATCC4525
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-keto-beta-methylvalerate + NH3 + NADH
? + H2O + NAD+
show the reaction diagram
-
as active as 2-ketoisocaproate, wild-type enzyme
-
-
?
2-keto-gamma-methylthiobutanoate + NH3 + NADH
L-Met + H2O + NAD+
show the reaction diagram
-
15% of the activity with 2-ketoisocaproate, wild-type enzyme
-
-
?
2-ketobutyrate + NH3 + NADH
L-2-aminobutyrate + H2O + NAD+
show the reaction diagram
2-ketocaproate + NH3 + NADH
L-2-aminohexanoate + H2O + NAD+
show the reaction diagram
-
69% of the activity with 2-ketoisocaproate
-
-
?
2-ketoisocaproate + NH3 + NADH
L-Ile + H2O + NAD+
show the reaction diagram
2-ketoisovalerate + NH3 + NADH
L-Val + H2O + NAD+
show the reaction diagram
-
150% of the activity with 2-ketoisocaproate
-
-
?
2-ketovalerate + NH3 + NADH
L-norvaline + H2O + NAD+
show the reaction diagram
2-oxo-3,3-dimethylbutanoate + NH3 + NADH
L-2-amino-3,3-dimethylbutanoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
2-oxo-4-methylselenobutyrate + NH3 + NADH
L-selenomethionine + H2O + NAD+
show the reaction diagram
-
-
-
?
2-oxobutanoate + NH3 + NADH
L-2-aminobutanoate + H2O + NAD+
show the reaction diagram
-
11% activity 2-oxobutanoate compared to 4-methyl-2-oxopentanoate
-
-
r
2-oxobutanoate + NH3 + NADH + H+
L-2-aminobutanoate + H2O + NAD+
show the reaction diagram
2-oxohexanoate + NH3 + NADH
L-norleucine + H2O + NAD+
show the reaction diagram
-
-
-
-
r
2-oxopentanoate + NH3 + NADH
L-norvaline + H2O + NAD+
show the reaction diagram
-
25% activity with 2-oxopentanoate compared to 4-methyl-2-oxopentanoate
-
-
r
2-oxophenylacetic acid + NH3 + NADH + H+
L-2-aminophenylacetic acid + H2O + NAD+
show the reaction diagram
-
-
-
-
r
3,3-dimethyl-2-oxobutanoate + NH3 + NADH
L-tert-Leu + H2O + NAD+
show the reaction diagram
3,3-dimethyl-2-oxobutanoate + NH3 + NADH + H+
L-tert-Leu + H2O + NAD+
show the reaction diagram
-
-
-
-
r
3-methyl-2-oxobutanoate + NH3 + NADH + H+
L-valine + H2O + NAD+
show the reaction diagram
-
-
-
-
r
3-methyl-2-oxopentanoate + NH3 + NADH
L-Ile + H2O + NAD+
show the reaction diagram
-
64% activity with 3-methyl-2-oxopentanoate compared to 4-methyl-2-oxopentanoate
-
-
r
4-methyl-2-oxo-5,5,5-trifluoropentanoate + NH3 + NADH
2-amino-4-methyl-5,5,5-trifluoropentanoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
4-methyl-2-oxopentanoate + NH3 + NADH
L-Leu + H2O + NAD+
show the reaction diagram
-
100% activity with 4-methyl-2-oxopentanoate
-
-
r
4-methyl-2-oxopentanoate + NH3 + NADH + H+
L-leucine + H2O + NAD+
show the reaction diagram
-
-
-
-
r
4-methylthio-2-oxobutyrate + NH3 + NADH
L-Met + H2O + NAD+
show the reaction diagram
-
19% activity with 4-methylthio-2-oxobutyrate compared to 4-methyl-2-oxopentanoate
-
-
r
alpha-keto beta-methylvalerate + NH3 + NADH + H+
L-isoleucine + H2O + NAD+
show the reaction diagram
yield: 95%
-
-
?
alpha-ketocaproate + NH3 + NADH + H+
L-norleucine + H2O + NAD+
show the reaction diagram
yield: 80%
-
-
?
alpha-ketoisocaproate + NH3 + NADH + H+
L-leucine + H2O + NAD+
show the reaction diagram
yield: 92.5%
-
-
?
alpha-ketoisovalerate + NH3 + NADH + H+
L-valine + H2O + NAD+
show the reaction diagram
yield: 90%
-
-
?
alpha-ketovalerate + NH3 + NADH + H+
L-norvaline + H2O + NAD+
show the reaction diagram
yield: 92%
-
-
?
L-2-aminobutanoate + H2O + NAD+
2-oxobutanoate + NH3 + NADH
show the reaction diagram
L-2-aminobutanoate + H2O + NAD+
2-oxopentanoate + NH3 + NADH
show the reaction diagram
L-2-aminobutyrate + H2O + NAD+
2-oxobutanoate + NH3 + NADH
show the reaction diagram
-
32% of the activity with L-Leu
-
-
?
L-Ala + H2O + NAD+
2-oxopropanoate + NH3 + NADH
show the reaction diagram
L-Ile + H2O + NAD+
3-methyl-2-oxopentanoate + NH3 + NADH
show the reaction diagram
L-isoleucine + H2O + NAD+
3-methyl-2-oxopentanoate + NH3 + NADH + H+
show the reaction diagram
L-Leu + H2O + 3-acetylpyridine-deamino-NAD+
4-methyl-2-oxopentanoate + NH3 + 3-acetylpyridine-deamino-NADH
show the reaction diagram
-
as effective as NAD+
-
-
?
L-Leu + H2O + 3-acetylpyridine-NAD+
4-methyl-2-oxopentanoate + NH3 + 3-acetylpyridine-NADH
show the reaction diagram
-
166% of the activity with NAD+
-
-
?
L-Leu + H2O + 3-pyridinealdehyde-NAD+
4-methyl-2-oxopentanoate + NH3 + 3-pyridinealdehyde-NADH
show the reaction diagram
-
19% of the activity with NAD+
-
-
?
L-Leu + H2O + deamino-NAD+
4-methyl-2-oxopentanoate + NH3 + deamino-NADH
show the reaction diagram
-
81% of the activity with NAD+
-
-
?
L-Leu + H2O + NAD+
4-methyl-2-oxopentanoate + NH3 + NADH
show the reaction diagram
L-Leu + H2O + thionicotinamide-NAD+
4-methyl-2-oxopentanoate + NH3 + thionicotinamide-NADH
show the reaction diagram
-
21% of the activity with NAD+
-
-
?
L-leucine + H2O + NAD+
4-methyl-2-oxopentanoate + NH3 + NADH + H+
show the reaction diagram
L-lysine + H2O + NAD+
6-amino-2-oxohexanoate + NH3 + NADH + H+
show the reaction diagram
-
69% of the activity with L-leucine
-
-
?
L-Met + H2O + NAD+
3-methylthio-2-oxobutanoate + NH3 + NADH
show the reaction diagram
L-Met + H2O + NAD+
4-methylthio-2-oxobutyrate + NH3 + NADH
show the reaction diagram
L-norleucine + H2O + NAD+
2-oxohexanoate + NH3 + NADH
show the reaction diagram
L-norleucine + H2O + NAD+
? + NH3 + NADH
show the reaction diagram
-
14% the activity with L-Leu, wild-type enzyme
-
-
?
L-norvaline + H2O + NAD+
2-ketovalerate + NH3 + NADH
show the reaction diagram
-
56% the activity with L-Leu, wild-type enzyme
-
-
?
L-norvaline + H2O + NAD+
2-oxopentanoate + NH3 + NADH
show the reaction diagram
L-Phe + H2O + NAD+
phenylpyruvate + NH3 + NADH
show the reaction diagram
-
no activity of wild-type enzyme activity with mutant enzymes A113G, A113G/V291L
-
-
?
L-S-methylcysteine + H2O + NAD+
3-methylthio-2-oxopropionate + NH3 + NADH
show the reaction diagram
-
19% of the activity with L-Leu
-
-
?
L-tert-Leu + H2O + NAD+
3,3-dimethyl-2-oxobutanoate + NH3 + NADH
show the reaction diagram
L-Val + H2O + NAD+
3-methyl-2-oxobutanoate + NH3 + NADH
show the reaction diagram
L-valine + H2O + NAD+
3-methyl-2-oxobutanoate + NH3 + NADH + H+
show the reaction diagram
phenylpyruvate + NH3 + NADH
L-Phe + H2O + NAD+
show the reaction diagram
-
15% of the activity with 2-ketoisocaproate, wild-type enzyme
-
-
?
S-methyl-L-cysteine + H2O + NAD+
3-methylthio-2-oxopropanoate + NH3 + NADH
show the reaction diagram
-
19% the activity with L-Leu
-
-
?
trimethylpyruvate + NH3 + NADH + H+
L-tert-leucine + H2O + NAD+
show the reaction diagram
trimethylpyruvic acid + NADH + NH3 + H+
L-tert-leucine + NAD+ + H2O
show the reaction diagram
-
-
-
-
-
additional information
?
-
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-Leu + H2O + NAD+
4-methyl-2-oxopentanoate + NH3 + NADH
show the reaction diagram
L-leucine + H2O + NAD+
4-methyl-2-oxopentanoate + NH3 + NADH + H+
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
-
wild-type enzyme is inactive, mutant enzyme D203A exhibits dual specificity for NAD+ and NADP+, mutant enzymes D203A/I204R and D203A/I204R/D210R show high affinity for NADP+
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CsCl
-
enzyme shows maximal activity in presence of more than 3 M salt
KCl
-
enzyme shows maximal activity in presence of more than 3 M salt
NaCl
-
enzyme shows maximal activity in presence of more than 3 M salt
RbCl
-
enzyme shows maximal activity in presence of more than 4 M salt
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-methyl-2-pentanone
-
competitive inhibition of wild-type enzyme, noncompetitive inhibition of mutant enzyme K80A
4-methylpentanoate
Ag+
-
1 mM, no residual activity
AgNO3
-
1 mM, complete
Cu(CH3COO)2
-
1 mM, 20% inhibition
D-2-aminobutanoate
-
-
D-alloisoleucine
-
-
Fe3+
-
1 mM, no residual activity
HgCl2
KCl
-
0.5 M, specific activity 133 U/mg
KCN
-
1 mM, 43% inhibition
L-Leu
-
competitive inhibition of the reductive amination of 4-methylthio-2-oxobutanoate
NaCl
-
0.5 M, specific activity 152 U/mg
NAD+
-
product inhibition
NH4+
-
inhibits oxidative deamination
p-mercuribenzoate
Pb(CH3COO)2
-
1 mM, 20% inhibition
pyridoxal 5'-phosphate
Sulfide
additional information
-
not inhibitory: EDTA, Ca2+, Mg2+
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4
2-ketoisocaproate
-
pH 9.5, 30°C
19
2-oxo-3,3-dimethylbutanoate
-
-
0.9 - 2.2
2-oxo-3-methylpentanoate
13
2-oxo-4-methylselenobutanoate
-
-
8.1
2-oxo-4-methylthiobutanoate
-
-
1.5 - 22.7
2-oxobutanoate
1.2 - 7
2-Oxohexanoate
0.88 - 25
2-Oxoisohexanoate
0.4 - 2.4
2-Oxopentanoate
2.6 - 217.7
3,3-dimethyl-2-oxobutanoate
2.41
3-acetylpyridine-deamino-NAD+
-
-
0.77
3-acetylpyridine-NAD+
-
-
1.4 - 4.4
3-methyl-2-oxobutanoate
0.38
3-methyl-2-oxopentanoate
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
2.1
3-methylthio-2-oxobutanoate
-
-
2.7
4-methyl-2-oxo-5,5,5-trifluoropentanoate
-
-
0.31 - 140
4-methyl-2-oxopentanoate
6.7
4-methylthio-2-oxobutanoate
-
-
3.8 - 69
alpha-keto-beta-methylvalerate
2 - 28
alpha-keto-caproate
0.88 - 30
alpha-keto-isocaproate
1.5 - 22
L-2-aminobutanoate
0.4 - 33
L-Ile
0.69 - 130
L-Leu
0.65 - 1.4
L-leucine
23 - 25
L-Met
1.27 - 24
L-norleucine
0.98 - 70
L-norvaline
31 - 66
L-Phe
1.8
L-tert-Leu
-
-
0.71 - 25
L-Val
0.015 - 17
NAD+
0.017 - 0.248
NADH
2.5 - 2.8
NADP+
220 - 330
NH3
13 - 750
NH4+
7.1 - 9.9
phenylpyruvate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.055 - 0.826
2-oxobutanoate
47.69
2-Oxopentanoate
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
10.4 - 40.4
3,3-dimethyl-2-oxobutanoate
27.63
3-methyl-2-oxopentanoate
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
99.38
4-methyl-2-oxopentanoate
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
37 - 280
alpha-keto-beta-methylvalerate
24 - 140
alpha-keto-caproate
29 - 280
alpha-keto-isocaproate
1.4 - 23
L-Ile
0.74 - 50
L-Leu
10
L-leucine
-
pH 10.5, 20°C
0.18 - 18
L-norleucine
0.29 - 13
L-norvaline
0.8 - 1.4
L-Phe
90
NAD+
-
wild-type enzyme
1.1 - 110
NADH
13
NADP+
-
mutant enzyme D203A/I204R and D203A/I204R/D210R
9.9 - 22
phenylpyruvate
additional information
additional information
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0025 - 0.1112
2-oxobutanoate
0.09 - 13
3,3-dimethyl-2-oxobutanoate
11 - 2800
NADH
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
140
4-methyl-2-pentanone
-
wild-type enzyme
20 - 33
4-methylpentanoate
9.4
D-2-aminobutanoate
-
inhibition of deamination of D-Leu
7.9
D-alloisoleucine
-
inhibition of deamination of D-Leu
2.2
D-Leu
-
inhibition of deamination of D-Leu
52.6
D-norleucine
-
inhibition of deamination of D-Leu
3
D-Norvaline
-
inhibition of deamination of D-Leu
10.8
D-Val
-
inhibition of deamination of D-Leu
1
L-Leu
-
competitive inhibition of the reductive amination of 4-methylthio-2-oxobutanoate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.074
-
strain DSM 3, pH 10.5, 25°C
0.88
pH 7.0, temperature not specified in the publication
2.6
-
wild-type, pH 7.5, 37°C
6.27
-
enzyme from cell-free extract, using L-Leu as substrate
8.88
-
mutant M374G, pH 7.5, 37°C
10.4
-
mutant M347G/Q358N, pH 7.5, 37°C
11.94
-
mutant Q358N, pH 7.5, 37°C
12.19
-
enzyme after Ni-NTA agaose gel purification, using L-Leu as substrate
53.8
-
purified enzyme, pH 10.5, 25°C
122
-
recombinant enzyme
133
-
substrate L-leucine, presence of 0.5 M KCl, pH 10.5, 25°C
152
-
substrate L-leucine, presence of 0.5 M NaCl, pH 10.5, 25°C
183
-
substrate L-leucine, pH 10.5, 25°C
320
-
pH not specified in the publication, temperature not specified in the publication
327
-
substrate L-leucine, pH 10.5, 60°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
-
reductive amination of 2-oxopentanoate and 3-methyl-2-oxobutanoate
8.6
-
reductive amination of 3-methyl-2-oxobutanoate and 2-oxopentanoate
8.8
-
reductive amination of 3-methyl-2-oxobutanoate
9 - 9.5
10.3
-
oxidative deamination of L-Leu in presence of 1.0 M NaCl
10.5 - 10.8
-
oxidative deamination of L-Leu and L-Val, phosphopyridoxylated enzyme
10.5
-
deamination of L-Leu
additional information
-
pH-optima for the chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 10
-
activity range, profile, overview
8 - 10
-
pH 8: about 30% of maximal activity, pH 10: about 70% of maximal activity, reductive amination of 2-ketoisocaproate
8.7 - 10.7
-
pH 8.7: about 65% of maximal activity, pH 10.7: about 45% of maximal activity, reductive amination of 2-oxo-4-methylselenobutanoate
9 - 10.5
-
pH 9: about 40% of maximal activity, pH 10.5: about 55% of maximal activity, oxidative deamination of L-Leu
9.5 - 11
-
-
10 - 11.5
-
pH 10.0: about 40% of maximal activity, pH 11.5: about 80% of maximal activity, oxidative deamination of L-Leu
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 37
more than 90% of maximum activity
0 - 50
-
highest activity at 50°C, 10% of maximal activity at 0°C
30 - 52
-
30°C: about 45% of maximal activity, 48-52°C: maximal activity
50 - 80
-
50°C: 37% of maximal activity, 80°C: 55% of maximal activity, oxidative amination of L-Leu
PDB
SCOP
CATH
UNIPROT
ORGANISM
Q7SIB4
Lysinibacillus sphaericus;
I0IJU1
Sporosarcina psychrophila;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000
-
2 * 40000, SDS-PAGE
41000
-
6 * 41000, SDS-PAGE
42000
-
4 * 42000, SDS-PAGE
44000
-
8 * 44000, SDS-PAGE
46903
-
x * 46903, calculation from nucleotide sequence
47000
-
6 * 47000, SDS-PAGE
49000
-
6 * 49000, SDS-PAGE
55000
-
6 * 55000, at 2.5 M NaCl, dissociates into trimers and dimers in a lower concentration of salts, cetyltrimethylammonium-bromide-PAGE
56000
-
6 * 56000, SDS-PAGE
82000
-
mutant enzyme LeuDEL4, gel filtration
245000
-
equilibrium sedimentation
280000
300000
-
gel filtration
310000
-
gel filtration, equilibrium sedimenation
313000
-
non-denaturing PAGE
320000
-
gel filtration
325000
-
gel filtration
330000
-
gel filtration
340000
350000
-
gel filtration
360000
additional information
-
MW of the chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase is 72000 Da, determined by gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homooctamer
octamer
additional information
-
catalytic triad consists of residues Lys 68, Lys 80 and Asp 115
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
homology modeling of structure and semirational engineering to increase the catalytic efficiency
-
crystallized by addition of ammonium sulfate
-
hanging drop method of vapour diffusion, using ammonium sulfate as the precipitant
-
crystals of the binary complex with 4-methyl-2-oxopentanoate, hanging-drop vapour-diffusion method using PEG 4000 as precipitant
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
-
purified enzyme, fully stable at 25°C
725737
5.4 - 10.3
-
55°C, 10 min, stable
349681
5.5 - 10
-
55°C, 5 min, stable
349662
5.6 - 9.8
-
25°C, 24 h, maximal loss of 12% of the activity
349661
6 - 11
-
55°C, 10 min, stable
349674
6 - 10
-
55°C, 10 min, stable
349680
6 - 10.9
-
50°C, 5 min, stable
349685
6.5 - 10.5
-
30 min, 50% loss of activity
349664
7 - 11.2
-
25°C, 30 min, wild-type enzyme and mutant enzymes K80A, K80R and K80Q
349691
7.5
-
70°C, 5 min, most stable at pH 7.5
349685
8 - 11
-
stable for at least 10 h
742176
9.5
-
mutant enzyme LeuDEL4 is stable
656566
9.5 - 10.5
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native enzyme is stable
656566
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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purified enzyme, fully stable at pH 5.0-11.0
52
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denaturation above
53
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unfolding temperature of mutant enzyme G78A
54
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pH 7.0-9.5, 60 min, chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase, stable
58
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pH 7.0-9.5, 60 min, chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase, loss of activity
76
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unfolding temperature of mutant enzyme G79A
79
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unfolding temperature of mutant enzyme K80R and K80Q
85
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5 min, substantial loss of activity
94
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10 min, 50% loss of activity, enzyme from spores
additional information
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme requires a high concentration of salt for stability
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mutant enzymes G77A and G78A show faster degradation than wild-type enzyme after incubation at 37°C for 15 h with trypsin or subtilisin at a protease-to-substrate ratio of 1:1. Wild-type enzyme and mutant enzyme G79A are degraded at almost the same rate
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the specific activity is increased nearly two times by Ni-NTA agarose gel purification
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-propanol
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2 M, 4°C, 5% loss of activity after 2 months
acetonitrile
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2 M, 4°C, 22% loss of activity after 3 d
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50% glycerol, stable for 1 year
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-20°C, stable for over 1 month
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30°C, 2 months, 50% loss of activity in presence of 2.5 M NaCl
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4°C, 5 mM tetrasodium EDTA solution in phosphate buffer, 0.1 M, pH 7.0, stable for at least 2 months
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4°C, 50 mM potassium phosphate, 1 mM dithioerythritol, pH 7.5, 15% loss of activity after 2 months
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4°C, buffer containing 0.02% azide, stable for more than 1 year
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4°C, buffer containing 0.02% sodium azide, stable for more than 1 year
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4°C, purified enzyme without any stabilizer, 1 month, barely changed activity
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4°C, stable for at least 1 month
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4°C, stable for over two years
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity extraction
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chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase
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mutant enzymes K68A and K68R
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native enzyme 48fold by ammonium sulfate fractionation, anion exchange and hydrophobic interaction chromatography , and gel filtration to over 95% purity
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Ni-NTA agarose gel chromatography
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one-step purification of recombinant enzyme
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recombinant enzyme
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a series of bifunctional enzyme complexes are produced by fusing leucine dehydrogenase and formate dehydrogenase with different peptide linkers, which are expressed in Escherichia coli, purified, and exhibit varied parental enzyme activities and varied L-tert leucine catalytic efficiency. The enzymatic reaction system for L-tert-leucine production and cofactor regeneration with suitable peptide linker is potentially more excellent than free enzymes with lower labor-cost on purification, better thermal stability and higher catalytic efficiency compared with the free coupling of parental enzymes
chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase
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cloned into Escherichia coli JM 109 with a vector plasmid pUC18
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expressed in Escherichia coli Rosetta 2(DE3)pLysS cells
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expression in Escherichia coli
expression in Escherichia coli C600
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overexpressed in Escherichia coli
production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system Escherichia coli[pIET98]
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recombinant expression in Escherichia coli strain SC16591
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
M347G
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251% of wild-type activity
M347G/Q358N
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400% of wild-type activity
M347N
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228% of wild-type activity
N262N
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121% of wild-type activity
N70F
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128% of wild-type activity
Q358N
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310% of wild-type activity
Q358T
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248% of wild-type activity; 257% of wild-type activity
A113G
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mutant enzyme with altered substrate specificity. 17.9fold decrease in turnover number for L-Leu, 1.2fold decrease in turnover-number for L-Ile, 13.8fold increase in turnover number of L-norleucine, 1.7fold decrease in turnover-number for L-norvaline, 3fold decrease in turnover number for alpha-keto-isocaproate, 1.2fold decrease in turnover number for alpha-ketocaproate, 1.3fold increase in turnover number for alpha-ketocaproate, 3.6fold decrease in Km-value for L-Leu, 3.3fold increase in Km-value for L-Ile, 1.1fold decrease in Km-value for L-norleucine, 3.5fold increase in Km-value for L-norvaline, 1.9fold increase in Km-value for alpha-keto-isocaproate, 2.5fold increase in Km-value for alpha-keto-beta-methylvalerate, 2.4fold decrease in Km-value for alpha-ketocaproate, 1.2fold increase in Km-value for NAD+, 1.2fold increase in Km-value for NADH as compared to wild-type enzyme. L-Ethionine and L-Phe are not substrates of the wild-type enzyme but are deaminated by mutant enzyme. Phenylpyruvate is not a substrate of the wild-type enzyme, but is aminated by mutant enzyme
A113G/V291L
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mutant enzyme with altered substrate specificity. 67.6fold decrease in turnover number for L-Leu, 20fold decrease in turnover-number for L-Ile, 2.2fold decrease in turnover number of L-norleucine, 44.8fold decrease in turnover-number for L-norvaline, 9.7fold decrease in turnover number for alpha-keto-isocaproate, 7.6fold decrease in turnover number for alpha-ketocaproate, 4.6fold decrease in turnover number for alpha-ketocaproate, 6.9fold increase in Km-value for L-Leu, 13.8fold increase in Km-value for L-Ile, 5.5fold increase in Km-value for L-norleucine, 9fold increase in Km-value for L-norvaline, 34fold increase in Km-value for alpha-keto-isocaproate, 18.2fold increase in Km-value for alpha-keto-beta-methylvalerate, 6fold increase in Km-value for alpha-ketocaproate, 4.4fold increase in Km-value for NAD+, 2fold decrease in Km-value for NADH as compared to wild-type enzyme. L-Ethionine and L-Phe are not substrates of the wild-type enzyme but are deaminated by mutant enzyme. Phenylpyruvate is not a substrate of the wild-type enzyme, but is aminated by mutant enzyme
G77A
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turnover numver in oxidative deamination of L-Leu is 36% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 6.3fold higher and the Km-value for NH4+ is 2.8fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme. Faster degradation than wild-type enzyme after incubation at 37°C for 15 h with trypsin or subtilisin at a protease-to-substrate ratio of 1:1
G78A
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turnover number in oxidative deamination of L-Leu is 5.4% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 8.8fold higher and the Km-value for NH4+ is 10fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme. Faster degradation than wild-type enzyme after incubation at 37°C for 15 h with trypsin or subtilisin at a protease-to-substrate ratio of 1:1
G79A
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turnover number in oxidative deamination of L-Leu is 40% of that of the wild-type enzyme. In reductive amination the turnover number is comparable to that of the wild-type enzyme. The Km-value for 2-oxoisohexanoate is 6.4fold higher and the Km-value for NH4+ is 3.9fold higher than that of the wild-type enzyme. Mutant enzyme shows lowered unfolding temperature compared with the wild-type enzyme
K68A
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nearly complete loss of activity in the oxidative deamination, marked increase in Km-values for both amino acid substrates and oxo acid substrates. An ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, is unobservable in mutant enzyme
K68R
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nearly complete loss of activity in the oxidative deamination. An ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, is unobservable in mutant enzyme
K80A
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markedly reduced activity in oxidative deamination, nearly 90% of the wild-type activity in reductive amination. Km-value for 2-oxoisohexanoate is 11fold higher than that of the wild-type enzyme, Km-value for L-Leu is lower than that of the wild-type enzyme
K80Q
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markedly reduced activity in oxidative deamination. Km-value for 2-oxoisohexanoate is 28fold higher than that of the wild-type enzyme, Km-value for L-Leu is about 3times larger than that of the wild-type enzyme
K80R
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markedly reduced activity in oxidative deamination, 0.6% of the wild-type activity in reductive amination, Km-value for L-Leu is lower than that of the wild-type enzyme
A43V/D124E
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mutant shows improved efficiency of L-tert-leucine synthesis, 5fold increase in catalyic efficiency compared to wild-type
LeuDEL4
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4 C-terminal amino acids deleted, mutant enzyme is a dimer
D203A
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dual specificity for NAD+ and NADP+
D203A/I204R
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high affinity for NADP+
D203A/I204R/D210R
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high affinity for NADP+
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
an efficient stereospecific enzymatic synthesis of L-valine, L-leucine, L-norvaline, L-norleucine and L-isoleucine from the corresponding alpha-keto acids by coupling the reactions catalysed by leucine dehydrogenase and glucose dehydrogenase/galactose mutarotase. Giving high yields of L-amino acids, the procedure is economical and easy to perform and to monitor at a synthetically useful scale (1-10 g)
drug development
synthesis
Show AA Sequence (687 entries)
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