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Literature summary for 1.1.1.41 extracted from

  • Tang, W.G.; Song, P.; Cao, Z.Y.; Wang, P.; Zhu, G.P.
    A unique homodimeric NAD+-linked isocitrate dehydrogenase from the smallest autotrophic eukaryote Ostreococcus tauri (2015), FASEB J., 29, 2462-2472.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
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

Protein Variants

Protein Variants Comment Organism
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
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
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

Inhibitors Comment Organism Structure
2-oxoglutarate strong inhibition at 10 mM Ostreococcus tauri
ATP strong inhibition at 10 mM Ostreococcus tauri
additional information no inhibition by NADPH and citrate Ostreococcus tauri
NADH comptitive with respect to NAD+ Ostreococcus tauri

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
0.0139
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri
0.067
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri
0.226
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri
0.265
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
0.738
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri
3.354
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
4.314
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri
20.65
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri

Localization

Localization Comment Organism GeneOntology No. Textmining
mitochondrion resticted to Ostreococcus tauri 5739
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ activates Ostreococcus tauri
Mn2+ activates Ostreococcus tauri

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
46700
-
-
Ostreococcus tauri
93000
-
about, sequence calculation Ostreococcus tauri
108000
-
recombinant His6-tagged enzyme, gel filtration Ostreococcus tauri

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
isocitrate + NAD+ Ostreococcus tauri
-
2-oxoglutarate + CO2 + NADH + H+
-
?

Organism

Organism UniProt Comment Textmining
Ostreococcus tauri
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3) by Co2+ affinity chromatography Ostreococcus tauri

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
DL-isocitrate + NAD+
-
Ostreococcus tauri 2-oxoglutarate + CO2 + NADH
-
?
DL-isocitrate + NADP+ very low activity Ostreococcus tauri 2-oxoglutarate + CO2 + NADPH
-
?
isocitrate + NAD+
-
Ostreococcus tauri 2-oxoglutarate + CO2 + NADH + H+
-
?

Subunits

Subunits Comment Organism
homodimer 2 * 47000-49000, recombinant His6-tagged enzyme, SDS-PAGE, 2 * 46700, about, sequence calculation Ostreococcus tauri
More comparative protein structure modeling using the mature OtIDH protein sequence, overview Ostreococcus tauri

Synonyms

Synonyms Comment Organism
homodimeric NAD+-linked isocitrate dehydrogenase
-
Ostreococcus tauri
NAD+-dependent isocitrate dehydrogenase
-
Ostreococcus tauri
NAD-IDH
-
Ostreococcus tauri
OtIDH
-
Ostreococcus tauri

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.9
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri
17
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
41
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri
46
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri
56
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri
59
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
80.9
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri
115
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.6
-
assay at Ostreococcus tauri

Cofactor

Cofactor Comment Organism Structure
additional information in the presence of Mn2+ and Mg2+, OtIDH displays 42fold and 51fold preference for NAD+ over NADP+, respectively Ostreococcus tauri
NAD+ specific for Ostreococcus tauri

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.14
-
NADH pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme Ostreococcus tauri

General Information

General Information Comment Organism
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
metabolism NAD-IDH is a key enzyme in the Krebs cycle Ostreococcus tauri
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]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.9
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri
5.1
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
10.7
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri
55.6
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri
261
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mg2+ Ostreococcus tauri
281
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R/M345H, with Mn2+ Ostreococcus tauri
434
-
NAD+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant wild-type enzyme, with Mn2+ Ostreococcus tauri
836
-
NADP+ pH 7.6, temperature not specified in the publication, His6-tagged recombinant mutant D344R, with Mn2+ Ostreococcus tauri