BRENDA - Enzyme Database

Identification of a novel trifunctional homoisocitrate dehydrogenase and modulation of the broad substrate specificity through site-directed mutagenesis

Miyazaki, K.; Biochem. Biophys. Res. Commun. 336, 596-602 (2005)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.87
DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli strain OM17
Deinococcus radiodurans
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.87
A80del
site-directed mutagenesis, the mutant shows altered substrate specificity preferring isocitrate to homoisocitrate, it is unable to oxidize 3-isopropylmalate, the specificity is similar to the enzyme from Thermus thermophilus
Deinococcus radiodurans
1.1.1.87
additional information
modulation of the broad substrate specificity of the trifunctional enzyme through site-directed mutagenesis, overview
Deinococcus radiodurans
1.1.1.87
R87T
site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
Deinococcus radiodurans
1.1.1.87
R87V
site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
additional information
deletion of A80, inability of mutant to use 3-isopropylmalate, isocitrate is 4fold preferred over homoisocitrate
Deinococcus radiodurans
1.1.1.286
R85V
increased ability to use 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
R87T
uses substrate homoisocitrate, but not substrates isocitrate or 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
R87V
uses substrate homoisocitrate, but not substrates isocitrate or 3-isopropylmalate
Deinococcus radiodurans
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.286
K+
absolute requirement, inhibitory above 0.4 M
Deinococcus radiodurans
1.1.1.286
Mn2+
inhibitory above 1 mM
Deinococcus radiodurans
1.1.1.286
NH4+
may substitute for K+, inhibitory above 0.2 M
Deinococcus radiodurans
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.87
0.211
-
homoisocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
0.291
-
isocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
0.465
-
isocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
0.819
-
homoisocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
1
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87V
Deinococcus radiodurans
1.1.1.87
1.33
-
3-isopropylmalate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
1.5
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87T
Deinococcus radiodurans
1.1.1.286
0.193
-
NAD+
30C, pH 7.8
Deinococcus radiodurans
1.1.1.286
0.211
-
homoisocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
0.291
-
isocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
0.465
-
isocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
0.819
-
homoisocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
1
-
homoisocitrate
30C, pH 7.8, mutant R87V
Deinococcus radiodurans
1.1.1.286
1.33
-
3-isopropylmalate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
1.5
-
homoisocitrate
30C, pH 7.8, mutant R87T
Deinococcus radiodurans
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.87
K+
required
Deinococcus radiodurans
1.1.1.87
Mn2+
required
Deinococcus radiodurans
1.1.1.286
K+
absolute requirement, inhibitory above 0.4 M
Deinococcus radiodurans
1.1.1.286
Mg2+
divalent cation required, Mg2+ is preferred over Mn2+
Deinococcus radiodurans
1.1.1.286
Mn2+
divalent cation required, Mg2+ is preferred over Mn2+, inhibitory above 1 mM
Deinococcus radiodurans
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.87
40000
-
4 * 40000, recombinant enzyme, SDS-PAGE
Deinococcus radiodurans
1.1.1.87
154000
-
recombinant enzyme, gel filtration
Deinococcus radiodurans
1.1.1.286
35727
-
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
1.1.1.286
35751
-
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
1.1.1.286
154000
-
gel filtration
Deinococcus radiodurans
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.87
Deinococcus radiodurans
-
-
-
1.1.1.286
Deinococcus radiodurans
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.87
recombinant enzyme from Escherichia coli strain OM17 by anion exchange and hydrophobic interaction chromatography, followed by another step of anion exchange chromatography
Deinococcus radiodurans
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.1.1.87
additional information
-
-
Deinococcus radiodurans
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.87
3-isopropylmalate + NAD+
-
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.87
homoisocitrate + NAD+
1.5fold preferred to isocitrate
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.87
isocitrate + NAD+
-
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.286
3-isopropylmalate + NAD+
at 0.1% of the rate with homoisocitrate
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
1.5fold preferred over isocitrate
660918
Deinococcus radiodurans
? + NADH
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
660918
Deinococcus radiodurans
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
isocitrate + NADP+
NAD+ is preferred over NADP+
660918
Deinococcus radiodurans
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.286
additional information
no substrate: NADP+
660918
Deinococcus radiodurans
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.87
tetramer
4 * 40000, recombinant enzyme, SDS-PAGE
Deinococcus radiodurans
1.1.1.286
tetramer
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
Temperature Optimum [C]
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.1.1.87
25
-
assay at
Deinococcus radiodurans
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.87
0.371
-
3-isopropylmalate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
5.46
-
homoisocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
10.9
-
isocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
20.8
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87V
Deinococcus radiodurans
1.1.1.87
27.8
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87T
Deinococcus radiodurans
1.1.1.87
42.8
-
isocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
46.2
-
homoisocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.286
0.371
-
3-isopropylmalate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
5.46
-
homoisocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
10.9
-
isocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
20.8
-
homoisocitrate
30C, pH 7.8, mutant R87V
Deinococcus radiodurans
1.1.1.286
27.8
-
homoisocitrate
30C, pH 7.8, mutant R87T
Deinococcus radiodurans
1.1.1.286
30.3
-
NAD+
30C, pH 7.8
Deinococcus radiodurans
1.1.1.286
42.8
-
isocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
46.2
-
homoisocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.87
7.8
-
assay at
Deinococcus radiodurans
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.87
NAD+
-
Deinococcus radiodurans
1.1.1.286
NAD+
-
Deinococcus radiodurans
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.87
DNA and amino acid sequence determination and analysis, overexpression in Escherichia coli strain OM17
Deinococcus radiodurans
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.87
NAD+
-
Deinococcus radiodurans
1.1.1.286
NAD+
-
Deinococcus radiodurans
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.87
A80del
site-directed mutagenesis, the mutant shows altered substrate specificity preferring isocitrate to homoisocitrate, it is unable to oxidize 3-isopropylmalate, the specificity is similar to the enzyme from Thermus thermophilus
Deinococcus radiodurans
1.1.1.87
additional information
modulation of the broad substrate specificity of the trifunctional enzyme through site-directed mutagenesis, overview
Deinococcus radiodurans
1.1.1.87
R87T
site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
Deinococcus radiodurans
1.1.1.87
R87V
site-directed mutagenesis, the mutant oxidizes homoisocitrate, but not isocitrate and 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
additional information
deletion of A80, inability of mutant to use 3-isopropylmalate, isocitrate is 4fold preferred over homoisocitrate
Deinococcus radiodurans
1.1.1.286
R85V
increased ability to use 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
R87T
uses substrate homoisocitrate, but not substrates isocitrate or 3-isopropylmalate
Deinococcus radiodurans
1.1.1.286
R87V
uses substrate homoisocitrate, but not substrates isocitrate or 3-isopropylmalate
Deinococcus radiodurans
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.286
K+
absolute requirement, inhibitory above 0.4 M
Deinococcus radiodurans
1.1.1.286
Mn2+
inhibitory above 1 mM
Deinococcus radiodurans
1.1.1.286
NH4+
may substitute for K+, inhibitory above 0.2 M
Deinococcus radiodurans
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.87
0.211
-
homoisocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
0.291
-
isocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
0.465
-
isocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
0.819
-
homoisocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
1
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87V
Deinococcus radiodurans
1.1.1.87
1.33
-
3-isopropylmalate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
1.5
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87T
Deinococcus radiodurans
1.1.1.286
0.193
-
NAD+
30C, pH 7.8
Deinococcus radiodurans
1.1.1.286
0.211
-
homoisocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
0.291
-
isocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
0.465
-
isocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
0.819
-
homoisocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
1
-
homoisocitrate
30C, pH 7.8, mutant R87V
Deinococcus radiodurans
1.1.1.286
1.33
-
3-isopropylmalate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
1.5
-
homoisocitrate
30C, pH 7.8, mutant R87T
Deinococcus radiodurans
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.87
K+
required
Deinococcus radiodurans
1.1.1.87
Mn2+
required
Deinococcus radiodurans
1.1.1.286
K+
absolute requirement, inhibitory above 0.4 M
Deinococcus radiodurans
1.1.1.286
Mg2+
divalent cation required, Mg2+ is preferred over Mn2+
Deinococcus radiodurans
1.1.1.286
Mn2+
divalent cation required, Mg2+ is preferred over Mn2+, inhibitory above 1 mM
Deinococcus radiodurans
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.87
40000
-
4 * 40000, recombinant enzyme, SDS-PAGE
Deinococcus radiodurans
1.1.1.87
154000
-
recombinant enzyme, gel filtration
Deinococcus radiodurans
1.1.1.286
35727
-
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
1.1.1.286
35751
-
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
1.1.1.286
154000
-
gel filtration
Deinococcus radiodurans
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.87
recombinant enzyme from Escherichia coli strain OM17 by anion exchange and hydrophobic interaction chromatography, followed by another step of anion exchange chromatography
Deinococcus radiodurans
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [mol/min/mg]
Specific Activity Maximum [mol/min/mg]
Commentary
Organism
1.1.1.87
additional information
-
-
Deinococcus radiodurans
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.87
3-isopropylmalate + NAD+
-
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.87
homoisocitrate + NAD+
1.5fold preferred to isocitrate
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.87
isocitrate + NAD+
-
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.286
3-isopropylmalate + NAD+
at 0.1% of the rate with homoisocitrate
660918
Deinococcus radiodurans
?
-
-
-
?
1.1.1.286
homoisocitrate + NAD+
1.5fold preferred over isocitrate
660918
Deinococcus radiodurans
? + NADH
-
-
-
?
1.1.1.286
isocitrate + NAD+
-
660918
Deinococcus radiodurans
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.286
isocitrate + NADP+
NAD+ is preferred over NADP+
660918
Deinococcus radiodurans
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.286
additional information
no substrate: NADP+
660918
Deinococcus radiodurans
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.87
tetramer
4 * 40000, recombinant enzyme, SDS-PAGE
Deinococcus radiodurans
1.1.1.286
tetramer
4 * 35751, mass spectrometry, 4 * 35727, calculated
Deinococcus radiodurans
Temperature Optimum [C] (protein specific)
EC Number
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
1.1.1.87
25
-
assay at
Deinococcus radiodurans
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.87
0.371
-
3-isopropylmalate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
5.46
-
homoisocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
10.9
-
isocitrate
pH 7.8, 25C, recombinant mutant A80del
Deinococcus radiodurans
1.1.1.87
20.8
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87V
Deinococcus radiodurans
1.1.1.87
27.8
-
homoisocitrate
pH 7.8, 25C, recombinant mutant R87T
Deinococcus radiodurans
1.1.1.87
42.8
-
isocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.87
46.2
-
homoisocitrate
pH 7.8, 25C, recombinant wild-type enzyme
Deinococcus radiodurans
1.1.1.286
0.371
-
3-isopropylmalate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
5.46
-
homoisocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
10.9
-
isocitrate
30C, pH 7.8, A80 deletion mutant
Deinococcus radiodurans
1.1.1.286
20.8
-
homoisocitrate
30C, pH 7.8, mutant R87V
Deinococcus radiodurans
1.1.1.286
27.8
-
homoisocitrate
30C, pH 7.8, mutant R87T
Deinococcus radiodurans
1.1.1.286
30.3
-
NAD+
30C, pH 7.8
Deinococcus radiodurans
1.1.1.286
42.8
-
isocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
1.1.1.286
46.2
-
homoisocitrate
30C, pH 7.8, wild-type
Deinococcus radiodurans
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.87
7.8
-
assay at
Deinococcus radiodurans