Cloned (Comment) | Organism |
---|---|
enzyme expression in Escherichia coli strain XL-1 Blue | Pseudomonas putida |
expressed in Escherichia coli XL1-Blue cells | Pseudomonas putida |
Crystallization (Comment) | Organism |
---|---|
hanging drop vapour diffusion method with 17-20% polyethylene glycol 1500, 0.1 M Tris-HCl, pH 7.1, 0.2 mM CaCl2 and 10 mM acetoacetate | Pseudomonas putida |
recombinant enzyme, hanging drop vapor diffusion method, 0.003 ml of HBDH, 10 mg/ml, is mixed with an equal volume of crystallization buffer containing 17-20% PEG 1500, 0.1 M Tris-HCl, pH 7.1, 0.2 mM CaCl2, and 10 mM acetoacetate, room temperature/22°C, three different crystal forms, X-ray diffraction structure determination and analysis at resolutions between 1.9 and 2.1 A | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
H141A | strongly decreased activity | Pseudomonas putida |
L149A | inactive | Pseudomonas putida |
Q133A | decreased activity | Pseudomonas putida |
Q91A | decreased activity | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.6 | - |
(R)-3-hydroxybutanoate | wild type enzyme | Pseudomonas putida | |
4 | - |
(R)-3-hydroxybutanoate | mutant enzyme H141A | Pseudomonas putida | |
51 | - |
(R)-3-hydroxybutanoate | mutant enzyme Q91A | Pseudomonas putida | |
70 | - |
(R)-3-hydroxybutanoate | mutant enzyme Q133A | Pseudomonas putida |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-3-hydroxybutanoate + NAD+ | Pseudomonas putida | - |
acetoacetate + NADH | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Pseudomonas putida | Q9AE70 | - |
- |
Purification (Comment) | Organism |
---|---|
charge-controlled hydrophobic chromatography and Sephadex G-100 gel filtration | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-3-hydroxybutanoate + NAD+ | - |
Pseudomonas putida | acetoacetate + NADH | - |
r | |
(R)-3-hydroxybutanoate + NAD+ | model of the binding mode of the substrate D-3-hydroxybutyrate, Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate, NAD+ binding increases the flexibility of the substrate-binding loop and shifts the equilibrium between the open and closed forms towards the closed form, overview | Pseudomonas putida | acetoacetate + NADH | - |
r | |
(R)-3-hydroxybutanoate + NAD+ | - |
Pseudomonas putida | acetoacetate + NADH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
tetramer | x-ray crystallography | Pseudomonas putida |
Synonyms | Comment | Organism |
---|---|---|
D-3-hydroxybutyrate dehydrogenase | - |
Pseudomonas putida |
HBDH | - |
Pseudomonas putida |
More | the enzyme belongs to the family of short-chain dehydrogenases/reductases | Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
13 | - |
(R)-3-hydroxybutanoate | mutant enzyme H141A | Pseudomonas putida | |
215 | - |
(R)-3-hydroxybutanoate | mutant enzyme Q133A | Pseudomonas putida | |
411 | - |
(R)-3-hydroxybutanoate | mutant enzyme Q91A | Pseudomonas putida | |
432 | - |
(R)-3-hydroxybutanoate | wild type enzyme | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Pseudomonas putida | |
NAD+ | the substrate-binding loop, residues 187-210, is partially disordered in several subunits, in both the presence and absence of NAD+, closed conformation in the complex structure with NAD+, interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change from open to closed, NAD+ binding increases the flexibility of the substrate-binding loop and shifts the equilibrium between the open and closed forms towards the closed form | Pseudomonas putida | |
NADH | - |
Pseudomonas putida |