Information on EC 1.4.1.9 - leucine dehydrogenase

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The expected taxonomic range for this enzyme is: Bacteria, 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
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
Metabolic pathways
-
-
Valine, leucine and isoleucine biosynthesis
-
-
Valine, leucine and isoleucine degradation
-
-
leucine metabolism
-
-
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
strain ATCC4525
-
-
Manually annotated by BRENDA team
strain MS-3
-
-
Manually annotated by BRENDA team
strain MS-3
-
-
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-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
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-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-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-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
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
-
-
?
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
AgNO3
-
1 mM, complete
Cu(CH3COO)2
-
1 mM, 20% inhibition
D-2-Aminobutanoate
-
-
D-alloisoleucine
-
-
HgCl2
KCN
-
1 mM, 43% inhibition
L-Leu
-
competitive inhibition of the reductive amination of 4-methylthio-2-oxobutanoate
Mn2+
-
inhibits oxidative deamination
NAD+
-
product inhibition
NH4+
-
inhibits oxidative deamination
p-mercuribenzoate
Pb(CH3COO)2
-
1 mM, 20% inhibition
pyridoxal 5'-phosphate
Sulfide
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 - 7.7
2-oxobutanoate
1.2 - 7
2-Oxohexanoate
0.88 - 25
2-Oxoisohexanoate
0.4 - 2.4
2-Oxopentanoate
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
L-leucine
-
pH 10.5, 20°C
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 - 0.12
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
47.69
2-Oxopentanoate
Lysinibacillus sphaericus
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
27.63
3-methyl-2-oxopentanoate
Lysinibacillus sphaericus
-
in 100 mM glycine-KCl-KOH buffer (pH 10), at 25°C
99.38
4-methyl-2-oxopentanoate
Lysinibacillus sphaericus
-
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
Sporosarcina psychrophila
-
pH 10.5, 20°C
0.18 - 18
L-norleucine
0.29 - 13
L-norvaline
0.8 - 1.4
L-Phe
90
NAD+
Thermoactinomyces intermedius
-
wild-type enzyme
13
NADP+
Thermoactinomyces intermedius
-
mutant enzyme D203A/I204R and D203A/I204R/D210R
9.9 - 22
phenylpyruvate
additional information
additional information
-
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
6.27
-
enzyme from cell-free extract, using L-Leu as substrate
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
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
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 OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
68 - 70
-
oxidative amination of L-Leu
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
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
?
-
x * 46903, calculation from nucleotide sequence
hexamer
homooctamer
octamer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
9.5 - 10.5
-
native enzyme is stable
656566
9.5
-
mutant enzyme LeuDEL4 is stable
656566
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
purified enzyme, fully stable at pH 5.0-11.0
52
-
denaturation above
53
-
unfolding temperature of mutant enzyme G78A
54
-
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
-
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
60
-
unfolding temperature of mutant enzyme G77A
76
-
unfolding temperature of mutant enzyme G79A
79
-
unfolding temperature of mutant enzyme K80R and K80Q
85
-
5 min, substantial loss of activity
94
-
10 min, 50% loss of activity, enzyme from spores
additional information
-
-
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme requires a high concentration of salt for stability
-
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
-
the specific activity is increased nearly two times by Ni-NTA agarose gel purification
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2-propanol
-
2 M, 4°C, 5% loss of activity after 2 months
acetonitrile
-
2 M, 4°C, 22% loss of activity after 3 d
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50% glycerol, stable for 1 year
-
-20°C, stable for over 1 month
-
30°C, 2 months, 50% loss of activity in presence of 2.5 M NaCl
-
4°C, 5 mM tetrasodium EDTA solution in phosphate buffer, 0.1 M, pH 7.0, stable for at least 2 months
-
4°C, 50 mM potassium phosphate, 1 mM dithioerythritol, pH 7.5, 15% loss of activity after 2 months
-
4°C, buffer containing 0.02% azide, stable for more than 1 year
-
4°C, buffer containing 0.02% sodium azide, stable for more than 1 year
-
4°C, purified enzyme without any stabilizer, 1 month, barely changed activity
-
4°C, stable for at least 1 month
-
4°C, stable for over two years
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
affinity extraction
-
chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase
-
mutant enzymes K68A and K68R
-
native enzyme 48fold by ammonium sulfate fractionation, anion exchange and hydrophobic interaction chromatography , and gel filtration to over 95% purity
-
Ni-NTA agarose gel chromatography
-
one-step purification of recombinant enzyme
-
recombinant enzyme
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase
-
cloned into Escherichia coli JM 109 with a vector plasmid pUC18
-
expressed in Escherichia coli Rosetta 2(DE3)pLysS cells
-
expression in Escherichia coli
expression in Escherichia coli C600
-
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
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
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)
synthesis
Show AA Sequence (629 entries)
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