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 (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 (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 | 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 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 | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-3-hydroxybutanoyl-CoA + NAD+ | Clostridium butyricum | - |
acetoacetyl-CoA + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Clostridium butyricum | C4IEM5 | - |
- |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(S)-3-hydroxybutanoyl-CoA + NAD+ | - |
Clostridium butyricum | acetoacetyl-CoA + NADH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
homodimer | SDS-PAGE and gel filtration | Clostridium butyricum |
Synonyms | Comment | Organism |
---|---|---|
(S)-3-hydroxybutyryl-CoA dehydrogenase | - |
Clostridium butyricum |
3-hydroxybutyryl-CoA dehydrogenase | - |
Clostridium butyricum |
CbHBD | - |
Clostridium butyricum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at, acetoacetyl-CoA reduction | Clostridium butyricum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at, acetoacetyl-CoA reduction | Clostridium butyricum |
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 | 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 |