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Literature summary for 1.1.1.35 extracted from

  • Kim, E.J.; Kim, J.; Ahn, J.W.; Kim, Y.J.; Chang, J.H.; Kim, K.J.
    Crystal structure of (S)-3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum and its mutations that enhance reaction kinetics (2014), J. Microbiol. Biotechnol., 24, 1636-1643.
    View publication on PubMed

Application

Application Comment Organism
biofuel production the highly efficient mutant enzyme K50A/K54A/L232Y can be useful for increasing the production rate of n-butanol in biofuel production Clostridium butyricum
synthesis the highly efficient mutant enzyme K50A/K54A/L232Y can be useful for increasing the production rate of n-butanol Clostridium butyricum

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain B834 Clostridium butyricum

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His6-tagged wild-type enzyme in apoform and in complex with substrates acetoacetyl-CoA and NAD+, hanging drop vapour diffusion method, mixing of 30 mg/ml protein in 40 mM Tris-HCl, pH 8.0, 1 mM DTT, with or without 20 mM NAD+, and 20 mM acetoacetyl-CoA, with reservoir solution containing 0.2 M Li2SO4, 0.1 M CAPS, pH 10.5, and 2 M ammonium sulfate, 22°C, 7 days, X-ray diffraction structure determination and analysis at 1.8-2.54 A resolution, molecular replacement and structure modeling Clostridium butyricum

Protein Variants

Protein Variants Comment Organism
K50A site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme Clostridium butyricum
K50A//L232Y site-directed mutagenesis, the mutant shows about 3fold increased activity compared to the wild-type enzyme Clostridium butyricum
K50A/K54 site-directed mutagenesis, the mutant shows about 3fold increased activity compared to the wild-type enzyme Clostridium butyricum
K50A/K54A/L232Y site-directed mutagenesis, the mutant shows about 5fold increased activity compared to the wild-type enzyme Clostridium butyricum
K54A site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme Clostridium butyricum
K54A/L232Y site-directed mutagenesis, the mutant shows about 4fold increased activity compared to the wild-type enzyme Clostridium butyricum
L232Z site-directed mutagenesis, the mutant shows about 2.5fold increased activity compared to the wild-type enzyme Clostridium butyricum
additional information structure-based protein engineering of CbHBD for increasing the production rate of n-butanol in biofuel production Clostridium butyricum

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics of recombinant wild-type and mutant enzymes, detailed overview Clostridium butyricum

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Clostridium butyricum C4IEM5
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain B834 by nickel affinity chromatography and gel filtration to about 95% purity Clostridium butyricum

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(S)-3-hydroxybutanoyl-CoA + NAD+
-
Clostridium butyricum acetoacetyl-CoA + NADH + H+
-
r

Subunits

Subunits Comment Organism
homodimer SDS-PAGE and gel filtration Clostridium butyricum

Synonyms

Synonyms Comment Organism
(S)-3-hydroxybutyryl-CoA dehydrogenase
-
Clostridium butyricum
3-hydroxybutyryl-CoA dehydrogenase
-
Clostridium butyricum
CbHBD
-
Clostridium butyricum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at, acetoacetyl-CoA reduction Clostridium butyricum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at, acetoacetyl-CoA reduction Clostridium butyricum

Cofactor

Cofactor Comment Organism Structure
NAD+ mode of cofactor binding, overview. The NAD+-binding site is located at the G-x-G-x-x-G nucleotide-binding motif, comprising residues Gly8-Ala9-Gly10-Thr11-Met12-Gly13. The hydroxyl groups of a phosphate moiety are hydrogen-bonded with the main chain nitrogen atoms of Thr11 and Met12. The nicotinamide and the two ribose rings of NAD+ are stabilized through hydrogen bond interactions mediated by the conserved Asp31, Glu90, Lys95, Asn115, Ser117, and Asn141 residues. The adenine moiety of NAD+ is positioned at the hydrophobic pocket formed by hydrophobic residues such as Leu7, Ile32, Ala88, Ile89, Ile94, and Ile98. One exception is Arg30, which assists the binding of the adenine moiety of NAD+ through a hydrogen bond Clostridium butyricum
NADH
-
Clostridium butyricum

General Information

General Information Comment Organism
metabolism the enzyme catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA Clostridium butyricum
additional information the adenosine diphosphate moiety of NAD+ is not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule Clostridium butyricum