Literature summary for 1.1.1.35 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.

Application

Application	Comment	Organism
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)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Cupriavidus necator	Q0KEY8	-	-
Cupriavidus necator ATCC 17699	Q0KEY8	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(S)-3-hydroxybutanoyl-CoA + NAD+	-	Cupriavidus necator	acetoacetyl-CoA + NADH + H+	-	r
(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
(S)-3-hydroxybutanoyl-CoA + NAD+	-	Cupriavidus necator ATCC 17699	acetoacetyl-CoA + NADH + H+	-	r
(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

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism	Structure
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
NADH	-	Cupriavidus necator

General Information

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