Cloned (Comment) | Organism |
---|---|
gene icd, phylogenetic analysis, recombinant expression of His6-tagged enzyme in Escherichia coli strain Rosetta (DE3) | Bifidobacterium longum subsp. infantis |
Protein Variants | Comment | Organism |
---|---|---|
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 |
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 |
R314D | site-directed mutagenesis | Bifidobacterium longum subsp. infantis |
R314D/H315I/T327A | site-directed mutagenesis | Bifidobacterium longum subsp. infantis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics analysis, overview | Bifidobacterium longum subsp. infantis | |
0.01945 | - |
NADP+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis | |
0.13 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
0.324 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
3.58 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | 14.3% activity compared to Mn2+ | Bifidobacterium longum subsp. infantis | |
Mg2+ | 69.2% activity compared to Mn2+ | Bifidobacterium longum subsp. infantis | |
Mn2+ | best activating cation | Bifidobacterium longum subsp. infantis | |
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] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
45000 | - |
- |
Bifidobacterium longum subsp. infantis |
83000 | - |
recombinant His6-tagged enzyme, gel filtration | Bifidobacterium longum subsp. infantis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
isocitrate + NADP+ | Bifidobacterium longum subsp. infantis | - |
2-oxoglutarate + CO2 + NADPH + H+ | - |
r | |
isocitrate + NADP+ | Bifidobacterium longum subsp. infantis ATCC 15697 | - |
2-oxoglutarate + CO2 + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bifidobacterium longum subsp. infantis | B7GQR3 | - |
- |
Bifidobacterium longum subsp. infantis ATCC 15697 | B7GQR3 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography | Bifidobacterium longum subsp. infantis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
isocitrate + NAD+ | engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41 | Bifidobacterium longum subsp. infantis | 2-oxoglutarate + CO2 + NADH + H+ | - |
r | |
isocitrate + NAD+ | engineered enzyme mutant D253A/S257K/K260Q/R314D/H315I/T327A, cf. EC 1.1.1.41 | Bifidobacterium longum subsp. infantis ATCC 15697 | 2-oxoglutarate + CO2 + NADH + H+ | - |
r | |
isocitrate + NADP+ | - |
Bifidobacterium longum subsp. infantis | 2-oxoglutarate + CO2 + NADPH + H+ | - |
r | |
isocitrate + NADP+ | - |
Bifidobacterium longum subsp. infantis ATCC 15697 | 2-oxoglutarate + CO2 + NADPH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 45000, recombinant His6-tagged enzyme, SDS-PAGE | Bifidobacterium longum subsp. infantis |
Synonyms | Comment | Organism |
---|---|---|
ICD | - |
Bifidobacterium longum subsp. infantis |
NADP+-dependent IDH | - |
Bifidobacterium longum subsp. infantis |
NADP-IDH | - |
Bifidobacterium longum subsp. infantis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
60 | - |
with Mn2+ | Bifidobacterium longum subsp. infantis |
65 | - |
with Mg2+ | Bifidobacterium longum subsp. infantis |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
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 Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.307 | - |
NADP+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
0.518 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
11.7 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis | |
36.4 | - |
NADP+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
with Mn2+ | Bifidobacterium longum subsp. infantis |
8 | - |
with Mg2+ | Bifidobacterium longum subsp. infantis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
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 | |
NADP+ | - |
Bifidobacterium longum subsp. infantis | |
NADPH | - |
Bifidobacterium longum subsp. infantis |
General Information | Comment | Organism |
---|---|---|
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 |
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.95 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
3 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis | |
4 | - |
NAD+ | pH 8.0, 25°C, recombinant mutant D253A/S257K/K260Q/R314D/H315I/T327A | Bifidobacterium longum subsp. infantis | |
1870 | - |
NAD+ | pH 8.0, 25°C, recombinant wild-type enzyme | Bifidobacterium longum subsp. infantis |