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Literature summary extracted from

  • Kim, J.; Chang, J.H.; Kim, K.J.
    Crystal structure and biochemical properties of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha (2014), Biochem. Biophys. Res. Commun., 448, 163-168.
    View publication on PubMed

Application

EC Number Application Comment Organism
1.1.1.35 biofuel production 3-hydroxybutyryl-CoA dehydrogenase is an enzyme involved in the synthesis of the biofuel n-butanol by converting acetoacetyl-CoA to 3-hydroxybutyryl-CoA, molecular mechanism of n-butanol biosynthesis, overview Cupriavidus necator

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.35 gene paaH1, recombinant expression of C-terminally His6-tagged wild-type or selenomethionine-labeled enzymes in Escherichia coli strain B834 Cupriavidus necator

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.35 purified recombinant enzyme in apoform, as selenomethionine-labeled enzyme, and in complex with substrates acetoacetyl-CoA and NAD+, hanging drop vapour diffusion method, mixing of 50 mg/ml wild-type protein or selenomethionine-labeled enzyme in 40 mM Tris-HCl, pH 8.0, 1 mM DTT, with reservoir solution containing 2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, and 0.2 M sodium chloride, 22°C, 7 days, X-ray diffraction structure determination and analysis at 2.42-2.7 A resolution, molecular replacement using the crystal structure of the apo-form of RePaaH1, and structure modeling Cupriavidus necator

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.35 K56A site-directed mutagenesis, the mutant shows about twofold increased activity compared to the wild-type enzyme Cupriavidus necator
1.1.1.35 N190A site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme Cupriavidus necator
1.1.1.35 R52A site-directed mutagenesis, the mutant shows highly decreased activity compared to the wild-type enzyme Cupriavidus necator
1.1.1.35 S119A site-directed mutagenesis, almost inactive mutant Cupriavidus necator

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.35 additional information
-
 additional information Michaelis-Menten kinetics Cupriavidus necator
1.1.1.35 0.01825
-
 acetoacetyl-CoA pH 8.0, 30°C, recombinant enzyme Cupriavidus necator

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+ Cupriavidus necator
-
 acetoacetyl-CoA + NADH + H+
-
 r
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+ Cupriavidus necator ATCC 17699
-
 acetoacetyl-CoA + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.35 Cupriavidus necator Q0KEY8
-
-
1.1.1.35 Cupriavidus necator ATCC 17699 Q0KEY8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.35 recombinant C-terminally His6-tagged wild-type or selenomethionine-labeled enzymes from Escherichia coli strain B834 by nickel affinity chromatography and gel filtration Cupriavidus necator

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+
-
 Cupriavidus necator acetoacetyl-CoA + NADH + H+
-
 r
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+ substrates binding structure analysis, overview. The acetoacetyl-CoA substrate is positioned within the deep cleft between the N-terminal domain and C-terminal domain. The acetoacetyl moiety is positioned near the conserved catalytic residues Ser119, His140, and Asn190 Cupriavidus necator acetoacetyl-CoA + NADH + H+
-
 r
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+
-
 Cupriavidus necator ATCC 17699 acetoacetyl-CoA + NADH + H+
-
 r
1.1.1.35 (S)-3-hydroxybutanoyl-CoA + NAD+ substrates binding structure analysis, overview. The acetoacetyl-CoA substrate is positioned within the deep cleft between the N-terminal domain and C-terminal domain. The acetoacetyl moiety is positioned near the conserved catalytic residues Ser119, His140, and Asn190 Cupriavidus necator ATCC 17699 acetoacetyl-CoA + NADH + H+
-
 r

Synonyms

EC Number Synonyms Comment Organism
1.1.1.35 (S)-3-hydroxybutyryl-CoA dehydrogenase
-
 Cupriavidus necator
1.1.1.35 PaaH1
-
 Cupriavidus necator
1.1.1.35 RePaaH1
-
 Cupriavidus necator

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.35 30
-
 assay at Cupriavidus necator

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.35 45500
-
 acetoacetyl-CoA pH 8.0, 30°C, recombinant enzyme Cupriavidus necator

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.35 8
-
 assay at Cupriavidus necator

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.35 NAD+ the NAD+ cofactor is bound to the N-terminal Rossmann fold, the NAD+-binding pocket is made up of 5 loops, enzyme binding structure analysis, overview Cupriavidus necator
1.1.1.35 NADH
-
 Cupriavidus necator

General Information

EC Number General Information Comment Organism
1.1.1.35 additional information conserved catalytic residues are Ser119, His140, and Asn190 Cupriavidus necator