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Literature summary extracted from
- Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B (2000), Biochemistry, 39, 4581-4589.
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 5.3.3.1 | crystals of Y30F, Y55F, and Y30F/Y55F are grown in the solution containing 1.0 M sodium acetate and 0.1 M ammonium acetate, pH 4.6 by hanging drop method of vapor diffusion at 22°C. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30 | Pseudomonas putida |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 5.3.3.1 | D99L | the turnover number for 5-androstene-3,17-dione is 96.5fold lower than that of the wild-type enzyme, the KM-value is 1.9fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 3.8 kcal/mol, at 25°C, pH 7.0 | Pseudomonas putida |
| 5.3.3.1 | Y14F | mutation results in a loss of conformational stability of 4.4 kcal/mol, at 25°C, pH 7.0 | Pseudomonas putida |
| 5.3.3.1 | Y30F | the turnover number for 5-androstene-3,17-dione is 1.2fold lower than that of the wild-type enzyme, the KM-value is 1.1fold lower than that of the wild-type enzyme | Pseudomonas putida |
| 5.3.3.1 | Y30F/D99L | the turnover number for 5-androstene-3,17-dione is 521.6fold lower than that of the wild-type enzyme, the KM-value is 1.47fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 6.5 kcal/mol, at 25°C, pH 7.0 | Pseudomonas putida |
| 5.3.3.1 | Y30F/Y55F | the turnover number for 5-androstene-3,17-dione is 1.99fold lower than that of the wild-type enzyme, the KM-value is comparable to that of the wild-type enzyme | Pseudomonas putida |
| 5.3.3.1 | Y55F | the turnover number for 5-androstene-3,17-dione is 6fold lower than that of the wild-type enzyme, the KM-value is 2.17fold lower than that of the wild-type enzyme. mutation results in a loss of conformational stability of 3.5 kcal/mol, at 25°C, pH 7.0. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30 | Pseudomonas putida |
| 5.3.3.1 | Y55F/D99L | the turnover number for 5-androstene-3,17-dione is 17692fold lower than that of the wild-type enzyme, the KM-value is 1.24fold lower than that of the wild-type enzyme. Mutation results in a loss of conformational stability of 7.9 kcal/mol, at 25°C, pH 7.0 | Pseudomonas putida |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 5.3.3.1 | 0.023 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y55F | Pseudomonas putida |  |
| 5.3.3.1 | 0.0258 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme D99L | Pseudomonas putida | |
| 5.3.3.1 | 0.0502 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F/Y55F | Pseudomonas putida | |
| 5.3.3.1 | 0.0552 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F | Pseudomonas putida | |
| 5.3.3.1 | 0.0619 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y55F/D99L | Pseudomonas putida | |
| 5.3.3.1 | 0.0735 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F/D99L | Pseudomonas putida | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 5.3.3.1 | Pseudomonas putida | - |
biotype B | - |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 5.3.3.1 | 5-Androstene-3,17-dione | the hydrogen-bond network which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site provides the structural support that is needed for the enzyme to maintain the active site geometry optimized for both function and stability | Pseudomonas putida | 4-Androstene-3,17-dione | - |
? | |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 5.3.3.1 | 1.2 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y55F/D99L | Pseudomonas putida | |
| 5.3.3.1 | 40.7 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F/D99L | Pseudomonas putida | |
| 5.3.3.1 | 220 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme D99L | Pseudomonas putida | |
| 5.3.3.1 | 3510 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y55F | Pseudomonas putida | |
| 5.3.3.1 | 10680 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F/Y55F | Pseudomonas putida | |
| 5.3.3.1 | 10700 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F/Y55F | Pseudomonas putida | |
| 5.3.3.1 | 17800 | - |
androstene-3,17-dione | 25°C, pH 7.0, mutant enzyme Y30F | Pseudomonas putida | |
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