BRENDA - Enzyme Database

Biochemical characterization and complete conversion of coenzyme specificity of isocitrate dehydrogenase from bifidobacterium longum

Huang, S.; Cheng, H.; Wang, P.; Zhu, G.; Int. J. Mol. Sci. 17, 296 (2016)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.1.1.42
gene icd, phylogenetic analysis, recombinant expression of His6-tagged enzyme in Escherichia coli strain Rosetta (DE3)
Bifidobacterium longum subsp. infantis
Engineering
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
D253A/S257K/K260Q/R314D/H315I/T327A
site-directed mutagenesis, the mutant shows a witch in cofactor specificity from NADP+ to NAD+
Bifidobacterium longum subsp. infantis
1.1.1.42
additional information
the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. The loss in NADP+-dependent activity might result from the removal of favorable interactions between Arg314 or His315 and the 2'-phosphate group
Bifidobacterium longum subsp. infantis
1.1.1.42
R314D
site-directed mutagenesis
Bifidobacterium longum subsp. infantis
1.1.1.42
R314D/H315I/T327A
site-directed mutagenesis
Bifidobacterium longum subsp. infantis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1.1.42
additional information
-
additional information
kinetics analysis, overview
Bifidobacterium longum subsp. infantis
1.1.1.42
0.01945
-
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
0.13
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
0.324
-
NADP+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
3.58
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Co2+
14.3% activity compared to Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
Mg2+
69.2% activity compared to Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
Mn2+
best activating cation
Bifidobacterium longum subsp. infantis
1.1.1.42
additional information
the enzyme is dependent on divalent cations, overview. No activity with Zn2+, Cu2+, Ca2+, and Li+,very low activity with K+, Na+, Ni2+, and Rb+
Bifidobacterium longum subsp. infantis
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
45000
-
-
Bifidobacterium longum subsp. infantis
1.1.1.42
83000
-
recombinant His6-tagged enzyme, gel filtration
Bifidobacterium longum subsp. infantis
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+
Bifidobacterium longum subsp. infantis
-
2-oxoglutarate + CO2 + NADPH + H+
-
-
r
1.1.1.42
isocitrate + NADP+
Bifidobacterium longum subsp. infantis ATCC 15697
-
2-oxoglutarate + CO2 + NADPH + H+
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.1.1.42
Bifidobacterium longum subsp. infantis
B7GQR3
-
-
1.1.1.42
Bifidobacterium longum subsp. infantis ATCC 15697
B7GQR3
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.1.1.42
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography
Bifidobacterium longum subsp. infantis
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+
engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41
740551
Bifidobacterium longum subsp. infantis
2-oxoglutarate + CO2 + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NAD+
engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41
740551
Bifidobacterium longum subsp. infantis ATCC 15697
2-oxoglutarate + CO2 + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
740551
Bifidobacterium longum subsp. infantis
2-oxoglutarate + CO2 + NADPH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
740551
Bifidobacterium longum subsp. infantis ATCC 15697
2-oxoglutarate + CO2 + NADPH + H+
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
1.1.1.42
homodimer
2 * 45000, recombinant His6-tagged enzyme, SDS-PAGE
Bifidobacterium longum subsp. infantis
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
60
-
with Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
65
-
with Mg2+
Bifidobacterium longum subsp. infantis
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.42
45
-
enzyme BlIDH retains 50% of maximal activity after incubation at 45°C for 20 min with either Mn2+ or Mg2+
Bifidobacterium longum subsp. infantis
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
1.1.1.42
0.307
-
NADP+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
0.518
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
11.7
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
36.4
-
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
7.5
-
with Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
8
-
with Mg2+
Bifidobacterium longum subsp. infantis
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
additional information
the enzyme shows a 567 and 193fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues by rational mutagenesis
Bifidobacterium longum subsp. infantis
1.1.1.42
NADP+
-
Bifidobacterium longum subsp. infantis
1.1.1.42
NADPH
-
Bifidobacterium longum subsp. infantis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
gene icd, phylogenetic analysis, recombinant expression of His6-tagged enzyme in Escherichia coli strain Rosetta (DE3)
Bifidobacterium longum subsp. infantis
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.1.1.42
additional information
the enzyme shows a 567 and 193fold preference for NADP+ over NAD+ in the presence of Mg2+ and Mn2+, respectively. The coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues by rational mutagenesis
Bifidobacterium longum subsp. infantis
1.1.1.42
NADP+
-
Bifidobacterium longum subsp. infantis
1.1.1.42
NADPH
-
Bifidobacterium longum subsp. infantis
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
1.1.1.42
D253A/S257K/K260Q/R314D/H315I/T327A
site-directed mutagenesis, the mutant shows a witch in cofactor specificity from NADP+ to NAD+
Bifidobacterium longum subsp. infantis
1.1.1.42
additional information
the coenzyme specificity of BlIDH can be completely reversed from NADP+ to NAD+ by a factor of 2387 by replacing six residues. The loss in NADP+-dependent activity might result from the removal of favorable interactions between Arg314 or His315 and the 2'-phosphate group
Bifidobacterium longum subsp. infantis
1.1.1.42
R314D
site-directed mutagenesis
Bifidobacterium longum subsp. infantis
1.1.1.42
R314D/H315I/T327A
site-directed mutagenesis
Bifidobacterium longum subsp. infantis
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
kinetics analysis, overview
Bifidobacterium longum subsp. infantis
1.1.1.42
0.01945
-
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
0.13
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
0.324
-
NADP+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
3.58
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.1.1.42
Co2+
14.3% activity compared to Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
Mg2+
69.2% activity compared to Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
Mn2+
best activating cation
Bifidobacterium longum subsp. infantis
1.1.1.42
additional information
the enzyme is dependent on divalent cations, overview. No activity with Zn2+, Cu2+, Ca2+, and Li+,very low activity with K+, Na+, Ni2+, and Rb+
Bifidobacterium longum subsp. infantis
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.1.1.42
45000
-
-
Bifidobacterium longum subsp. infantis
1.1.1.42
83000
-
recombinant His6-tagged enzyme, gel filtration
Bifidobacterium longum subsp. infantis
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+
Bifidobacterium longum subsp. infantis
-
2-oxoglutarate + CO2 + NADPH + H+
-
-
r
1.1.1.42
isocitrate + NADP+
Bifidobacterium longum subsp. infantis ATCC 15697
-
2-oxoglutarate + CO2 + NADPH + H+
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.1.1.42
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography
Bifidobacterium longum subsp. infantis
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+
engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41
740551
Bifidobacterium longum subsp. infantis
2-oxoglutarate + CO2 + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NAD+
engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41
740551
Bifidobacterium longum subsp. infantis ATCC 15697
2-oxoglutarate + CO2 + NADH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
740551
Bifidobacterium longum subsp. infantis
2-oxoglutarate + CO2 + NADPH + H+
-
-
-
r
1.1.1.42
isocitrate + NADP+
-
740551
Bifidobacterium longum subsp. infantis ATCC 15697
2-oxoglutarate + CO2 + NADPH + H+
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.1.1.42
homodimer
2 * 45000, recombinant His6-tagged enzyme, SDS-PAGE
Bifidobacterium longum subsp. infantis
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.1.1.42
60
-
with Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
65
-
with Mg2+
Bifidobacterium longum subsp. infantis
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
1.1.1.42
45
-
enzyme BlIDH retains 50% of maximal activity after incubation at 45°C for 20 min with either Mn2+ or Mg2+
Bifidobacterium longum subsp. infantis
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
0.307
-
NADP+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
0.518
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
11.7
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
36.4
-
NADP+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.1.1.42
7.5
-
with Mn2+
Bifidobacterium longum subsp. infantis
1.1.1.42
8
-
with Mg2+
Bifidobacterium longum subsp. infantis
General Information
EC Number
General Information
Commentary
Organism
1.1.1.42
evolution
based on the phylogenetic analysis, IDHs can be divided into three subfamilies: Type I IDHs, Type II IDHs and monomeric IDHs. The enzyme BlIDH from Bifidobacterium longum belongs to the type II subfamily. NAD+ use is an ancestral trait and NADP+ use by bacterial IDHs arose on or about the time that eukaryotic mitochondria first appeared, some 3.5 billion years ago
Bifidobacterium longum subsp. infantis
1.1.1.42
physiological function
NADP+-dependent IDH generates NADPH, which provides the reducing power for biosynthesis, maintains the redox state of the cell, and takes part in CO2 assimilation
Bifidobacterium longum subsp. infantis
General Information (protein specific)
EC Number
General Information
Commentary
Organism
1.1.1.42
evolution
based on the phylogenetic analysis, IDHs can be divided into three subfamilies: Type I IDHs, Type II IDHs and monomeric IDHs. The enzyme BlIDH from Bifidobacterium longum belongs to the type II subfamily. NAD+ use is an ancestral trait and NADP+ use by bacterial IDHs arose on or about the time that eukaryotic mitochondria first appeared, some 3.5 billion years ago
Bifidobacterium longum subsp. infantis
1.1.1.42
physiological function
NADP+-dependent IDH generates NADPH, which provides the reducing power for biosynthesis, maintains the redox state of the cell, and takes part in CO2 assimilation
Bifidobacterium longum subsp. infantis
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
0.95
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
3
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
4
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
1870
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
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
0.95
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
3
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis
1.1.1.42
4
-
NAD+
pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A
Bifidobacterium longum subsp. infantis
1.1.1.42
1870
-
NAD+
pH 8.0, 25°C, recombinant wild-type enzyme
Bifidobacterium longum subsp. infantis