BRENDA - Enzyme Database

NADP+-specific isocitrate dehydrogenase from oleaginous yeast Yarrowia lipolytica CLIB122: biochemical characterization and coenzyme sites evaluation

Li, X.; Wang, P.; Ge, Y.; Wang, W.; Abbas, A.; Zhu, G.; Appl. Biochem. Biotechnol. 171, 403-416 (2013)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.42
idh2, DNA and amino acid sequence determination and anaysis, phylogenetic analysis, overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3)
Yarrowia lipolytica
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
R322D
site-directed mutagenesis, the mutant shows an 41fold higher Km for NADP+ than the wild-type enzyme, and it shows NAD+-dependent activity in contrast to the wild-type enzyme
Yarrowia lipolytica
1.1.1.42
R322D/H323I
site-directed mutagenesis, inactive mutant
Yarrowia lipolytica
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
additional information
-
additional information
kinetic analysis
Yarrowia lipolytica
1.1.1.42
0.031
-
NADP+
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.058
-
NADP+
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.059
-
isocitrate
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.12
-
isocitrate
pH 7.5, 25°C, recombinant enzyme, with Mn2+
Yarrowia lipolytica
1.1.1.42
47
-
NAD+
pH 7.5, 25°C, recombinant enzyme mutant R322D
Yarrowia lipolytica
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Co2+
activates to 43% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
Li+
activates to 24% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
Mg2+
most favorable cofactor
Yarrowia lipolytica
1.1.1.42
Mn2+
activates to 60% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
additional information
the enzyme is absolutely divalent cation dependent, no or poor activation by Ca2+, Zn2+, K+, Na+, and Rb+
Yarrowia lipolytica
1.1.1.42
Ni2+
activates to 10% of the activity with Mg2+
Yarrowia lipolytica
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
45000
-
2 * 45000, recombinant enzyme, SDS-PAGE
Yarrowia lipolytica
1.1.1.42
81300
-
recombinant enzyme, gel filtration
Yarrowia lipolytica
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.42
isocitrate + NADP+
Yarrowia lipolytica
-
2-oxoglutarate + NADPH + H+
-
-
r
1.1.1.42
isocitrate + NADP+
Yarrowia lipolytica CLIB 122
-
2-oxoglutarate + NADPH + H+
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.42
Yarrowia lipolytica
Q6C2Y4
-
-
1.1.1.42
Yarrowia lipolytica CLIB 122
Q6C2Y4
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.42
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) b metal affinity chromatography
Yarrowia lipolytica
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.42
isocitrate + NAD+
low activity with mutant enzyme R322D, no activity with the wild-type enzyme
739874
Yarrowia lipolytica
2-oxoglutarate + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NAD+
low activity with mutant enzyme R322D, no activity with the wild-type enzyme
739874
Yarrowia lipolytica CLIB 122
2-oxoglutarate + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
739874
Yarrowia lipolytica
2-oxoglutarate + NADPH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
739874
Yarrowia lipolytica CLIB 122
2-oxoglutarate + NADPH + H+
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
homodimer
2 * 45000, recombinant enzyme, SDS-PAGE
Yarrowia lipolytica
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
55
-
recombinant enzyme
Yarrowia lipolytica
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.42
45
55
purified recombinant enzyme, stable up to 45°C, enzyme activity drops quickly above this temperature, incubation at 50°C for 20 min causes a 40% loss of activity, while incubation at 55°C almost inactivates the enzyme
Yarrowia lipolytica
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.38
-
NAD+
pH 8.5, 55°C, recombinant mutant R322D
Yarrowia lipolytica
1.1.1.42
4.24
-
NADP+
pH 7.5, 25°C, recombinant mutant R322D, with Mg2+
Yarrowia lipolytica
1.1.1.42
72
-
NADP+
pH 7.5, 25°C, recombinant wild-type enzyme, with Mg2+
Yarrowia lipolytica
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
8.5
-
recombinant enzyme
Yarrowia lipolytica
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
additional information
enzyme YlIDP is absolutely dependent on NADP+ and shows no NAD-dependent activity
Yarrowia lipolytica
1.1.1.42
NADP+
-
Yarrowia lipolytica
1.1.1.42
NADPH
-
Yarrowia lipolytica
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
idh2, DNA and amino acid sequence determination and anaysis, phylogenetic analysis, overexpression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3)
Yarrowia lipolytica
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
additional information
enzyme YlIDP is absolutely dependent on NADP+ and shows no NAD-dependent activity
Yarrowia lipolytica
1.1.1.42
NADP+
-
Yarrowia lipolytica
1.1.1.42
NADPH
-
Yarrowia lipolytica
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
R322D
site-directed mutagenesis, the mutant shows an 41fold higher Km for NADP+ than the wild-type enzyme, and it shows NAD+-dependent activity in contrast to the wild-type enzyme
Yarrowia lipolytica
1.1.1.42
R322D/H323I
site-directed mutagenesis, inactive mutant
Yarrowia lipolytica
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
additional information
-
additional information
kinetic analysis
Yarrowia lipolytica
1.1.1.42
0.031
-
NADP+
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.058
-
NADP+
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.059
-
isocitrate
pH 7.5, 25°C, recombinant enzyme, with Mg2+
Yarrowia lipolytica
1.1.1.42
0.12
-
isocitrate
pH 7.5, 25°C, recombinant enzyme, with Mn2+
Yarrowia lipolytica
1.1.1.42
47
-
NAD+
pH 7.5, 25°C, recombinant enzyme mutant R322D
Yarrowia lipolytica
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Co2+
activates to 43% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
Li+
activates to 24% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
Mg2+
most favorable cofactor
Yarrowia lipolytica
1.1.1.42
Mn2+
activates to 60% of the activity with Mg2+
Yarrowia lipolytica
1.1.1.42
additional information
the enzyme is absolutely divalent cation dependent, no or poor activation by Ca2+, Zn2+, K+, Na+, and Rb+
Yarrowia lipolytica
1.1.1.42
Ni2+
activates to 10% of the activity with Mg2+
Yarrowia lipolytica
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
45000
-
2 * 45000, recombinant enzyme, SDS-PAGE
Yarrowia lipolytica
1.1.1.42
81300
-
recombinant enzyme, gel filtration
Yarrowia lipolytica
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.42
isocitrate + NADP+
Yarrowia lipolytica
-
2-oxoglutarate + NADPH + H+
-
-
r
1.1.1.42
isocitrate + NADP+
Yarrowia lipolytica CLIB 122
-
2-oxoglutarate + NADPH + H+
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) b metal affinity chromatography
Yarrowia lipolytica
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.42
isocitrate + NAD+
low activity with mutant enzyme R322D, no activity with the wild-type enzyme
739874
Yarrowia lipolytica
2-oxoglutarate + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NAD+
low activity with mutant enzyme R322D, no activity with the wild-type enzyme
739874
Yarrowia lipolytica CLIB 122
2-oxoglutarate + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
739874
Yarrowia lipolytica
2-oxoglutarate + NADPH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
739874
Yarrowia lipolytica CLIB 122
2-oxoglutarate + NADPH + H+
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
homodimer
2 * 45000, recombinant enzyme, SDS-PAGE
Yarrowia lipolytica
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
55
-
recombinant enzyme
Yarrowia lipolytica
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.42
45
55
purified recombinant enzyme, stable up to 45°C, enzyme activity drops quickly above this temperature, incubation at 50°C for 20 min causes a 40% loss of activity, while incubation at 55°C almost inactivates the enzyme
Yarrowia lipolytica
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.38
-
NAD+
pH 8.5, 55°C, recombinant mutant R322D
Yarrowia lipolytica
1.1.1.42
4.24
-
NADP+
pH 7.5, 25°C, recombinant mutant R322D, with Mg2+
Yarrowia lipolytica
1.1.1.42
72
-
NADP+
pH 7.5, 25°C, recombinant wild-type enzyme, with Mg2+
Yarrowia lipolytica
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
8.5
-
recombinant enzyme
Yarrowia lipolytica
General Information
EC Number
General Information
Commentary
Organism
1.1.1.42
evolution
phylogenetic analysis and evolutionary relationships between YlIDP and IDPs from other yeasts or yeast-related species
Yarrowia lipolytica
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.1.1.42
evolution
phylogenetic analysis and evolutionary relationships between YlIDP and IDPs from other yeasts or yeast-related species
Yarrowia lipolytica
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.42
1.75
-
NADP+
pH 7.5, 25°C, recombinant mutant R322D, with Mg2+
Yarrowia lipolytica
1.1.1.42
1220
-
NADP+
pH 7.5, 25°C, recombinant wild-type enzyme, with Mg2+
Yarrowia lipolytica
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.42
1.75
-
NADP+
pH 7.5, 25°C, recombinant mutant R322D, with Mg2+
Yarrowia lipolytica
1.1.1.42
1220
-
NADP+
pH 7.5, 25°C, recombinant wild-type enzyme, with Mg2+
Yarrowia lipolytica