BRENDA - Enzyme Database

A unique homodimeric NAD+-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri

Tang, W.G.; Song, P.; Cao, Z.Y.; Wang, P.; Zhu, G.P.; FASEB J. 29, 2462-2472 (2015)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.41
DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3)
Ostreococcus tauri
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
D344R
site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, and the mutant displays a 15fold preference for NADP+ over NAD+ compared to the wild-type enzyme
Ostreococcus tauri
1.1.1.41
D344R/M345H
site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, the mutant displays a 72fold preference for NADP+ over NAD+ compared to the wild-type enzyme
Ostreococcus tauri
1.1.1.41
additional information
the enzyme's coenzyme specificity can be completely converted from NAD+ (ancient trait) to NADP+ (adaptive trait) by rational mutagenesis based on the evolutionary trace. Residues D344 and M345 are the determinants of NAD+ specificity
Ostreococcus tauri
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.41
2-oxoglutarate
strong inhibition at 10 mM
Ostreococcus tauri
1.1.1.41
ATP
strong inhibition at 10 mM
Ostreococcus tauri
1.1.1.41
additional information
no inhibition by NADPH and citrate
Ostreococcus tauri
1.1.1.41
NADH
comptitive with respect to NAD+
Ostreococcus tauri
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
additional information
-
additional information
OtIDH exhibits a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric enzyme homologues, and a remarkably high affinity for isocitrate unlike other heterooligomeric enzyme homologues
Ostreococcus tauri
1.1.1.41
0.0139
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
0.067
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
0.226
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
0.265
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
0.738
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
3.354
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
4.314
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
20.65
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.1.1.41
mitochondrion
resticted to
Ostreococcus tauri
5739
-
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.41
Mg2+
activates
Ostreococcus tauri
1.1.1.41
Mn2+
activates
Ostreococcus tauri
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
46700
-
-
Ostreococcus tauri
1.1.1.41
93000
-
about, sequence calculation
Ostreococcus tauri
1.1.1.41
108000
-
recombinant His6-tagged enzyme, gel filtration
Ostreococcus tauri
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.41
isocitrate + NAD+
Ostreococcus tauri
-
2-oxoglutarate + CO2 + NADH
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.41
Ostreococcus tauri
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.41
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography
Ostreococcus tauri
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
DL-isocitrate + NAD+
-
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.41
DL-isocitrate + NADP+
very low activity
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.41
isocitrate + NAD+
-
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADH
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.41
homodimer
2 * 47000-49000, recombinant His6-tagged enzyme, SDS-PAGE, 2 * 46700, about, sequence calculation
Ostreococcus tauri
1.1.1.41
More
comparative protein structure modeling using the mature OtIDH protein sequence, overview
Ostreococcus tauri
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.41
3.9
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
17
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
41
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
46
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
56
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
59
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
80.9
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
115
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
7.6
-
assay at
Ostreococcus tauri
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
additional information
in the presence of Mn2+ and Mg2+, OtIDH displays 42fold and 51fold preference for NAD+ over NADP+, respectively
Ostreococcus tauri
1.1.1.41
NAD+
specific for
Ostreococcus tauri
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
1.1.1.41
0.14
-
NADH
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme
Ostreococcus tauri
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
DNA and amino acid sequence determination and analysis, phylogenetic tree, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3)
Ostreococcus tauri
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.41
additional information
in the presence of Mn2+ and Mg2+, OtIDH displays 42fold and 51fold preference for NAD+ over NADP+, respectively
Ostreococcus tauri
1.1.1.41
NAD+
specific for
Ostreococcus tauri
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.41
D344R
site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, and the mutant displays a 15fold preference for NADP+ over NAD+ compared to the wild-type enzyme
Ostreococcus tauri
1.1.1.41
D344R/M345H
site-directed mutagenesis, the mutant enzyme retains their native dimeric structure, the mutant displays a 72fold preference for NADP+ over NAD+ compared to the wild-type enzyme
Ostreococcus tauri
1.1.1.41
additional information
the enzyme's coenzyme specificity can be completely converted from NAD+ (ancient trait) to NADP+ (adaptive trait) by rational mutagenesis based on the evolutionary trace. Residues D344 and M345 are the determinants of NAD+ specificity
Ostreococcus tauri
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.1.1.41
2-oxoglutarate
strong inhibition at 10 mM
Ostreococcus tauri
1.1.1.41
ATP
strong inhibition at 10 mM
Ostreococcus tauri
1.1.1.41
additional information
no inhibition by NADPH and citrate
Ostreococcus tauri
1.1.1.41
NADH
comptitive with respect to NAD+
Ostreococcus tauri
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
1.1.1.41
0.14
-
NADH
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme
Ostreococcus tauri
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.41
additional information
-
additional information
OtIDH exhibits a sigmoidal kinetic behavior in response to isocitrate unlike other homodimeric enzyme homologues, and a remarkably high affinity for isocitrate unlike other heterooligomeric enzyme homologues
Ostreococcus tauri
1.1.1.41
0.0139
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
0.067
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
0.226
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
0.265
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
0.738
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
3.354
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
4.314
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
20.65
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.1.1.41
mitochondrion
resticted to
Ostreococcus tauri
5739
-
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.41
Mg2+
activates
Ostreococcus tauri
1.1.1.41
Mn2+
activates
Ostreococcus tauri
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.41
46700
-
-
Ostreococcus tauri
1.1.1.41
93000
-
about, sequence calculation
Ostreococcus tauri
1.1.1.41
108000
-
recombinant His6-tagged enzyme, gel filtration
Ostreococcus tauri
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.1.1.41
isocitrate + NAD+
Ostreococcus tauri
-
2-oxoglutarate + CO2 + NADH
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.41
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography
Ostreococcus tauri
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.1.1.41
DL-isocitrate + NAD+
-
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADH
-
-
-
?
1.1.1.41
DL-isocitrate + NADP+
very low activity
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADPH
-
-
-
?
1.1.1.41
isocitrate + NAD+
-
740425
Ostreococcus tauri
2-oxoglutarate + CO2 + NADH
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.41
homodimer
2 * 47000-49000, recombinant His6-tagged enzyme, SDS-PAGE, 2 * 46700, about, sequence calculation
Ostreococcus tauri
1.1.1.41
More
comparative protein structure modeling using the mature OtIDH protein sequence, overview
Ostreococcus tauri
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.41
3.9
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
17
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
41
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
46
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
56
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
59
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
80.9
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
115
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.41
7.6
-
assay at
Ostreococcus tauri
General Information
EC Number
General Information
Commentary
Organism
1.1.1.41
evolution
OtIDH may be an ancestral form of type II IDHs (all other reported members are NADP+-linked enzymes) and may have evolved into NADP+-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation
Ostreococcus tauri
1.1.1.41
metabolism
NAD-IDH is a key enzyme in the Krebs cycle
Ostreococcus tauri
1.1.1.41
additional information
structure homology modelling, overview. Residue Asp344 is clearly a major determinant of coenzyme specificity in OtIDH and residue Met345 plays critical role in NAD+ recognition and binding
Ostreococcus tauri
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.1.1.41
evolution
OtIDH may be an ancestral form of type II IDHs (all other reported members are NADP+-linked enzymes) and may have evolved into NADP+-dependent IDH for adaptation to the increased demand of NADPH under carbon starvation
Ostreococcus tauri
1.1.1.41
metabolism
NAD-IDH is a key enzyme in the Krebs cycle
Ostreococcus tauri
1.1.1.41
additional information
structure homology modelling, overview. Residue Asp344 is clearly a major determinant of coenzyme specificity in OtIDH and residue Met345 plays critical role in NAD+ recognition and binding
Ostreococcus tauri
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
1.1.1.41
3.9
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
5.1
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
10.7
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
55.6
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
261
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
281
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
434
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
836
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
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
1.1.1.41
3.9
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
5.1
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
10.7
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
55.6
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri
1.1.1.41
261
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+
Ostreococcus tauri
1.1.1.41
281
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+
Ostreococcus tauri
1.1.1.41
434
-
NAD+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+
Ostreococcus tauri
1.1.1.41
836
-
NADP+
pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+
Ostreococcus tauri