Cloned (Comment) | Organism |
---|---|
wild-type and mutant enzymes A460I, F464I and A460I/F464I | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
A460I | activity with pyruvate is higher than wild-type activity, activity with 2-oxobutanoic acid is 2.3fold higher than wild-type activity, activity with 2-oxopentanoic acid is 1.1fold lower than wild-type activity, activity with 2-oxohexanoic acid is 3.3fold higher than wild-type activity, activity with 3-methyl-2-oxobutanoic acid is 1.3fold higher than wild-type activity, activity with 3-methyl-2-oxopentanoic acid is 1.1fold lower than wild-type activity, activity with 2-oxo-4-methylpentanoic acid is 1.3fold higher than wild-type activity, activity with 4,4-dimethyl-2-oxopentanoic acid is identical to wild-type activity, activity with 2-oxo-4-methylhexanoic acid is 2.2fold higher than wild-type activity, activity with benzoylformate is 22.5fold lower than wild-type activity, activity with 2-oxo-3-phenylpropanoic acid is identical to wild-type activity, activity with 2-oxo-4-phenylbutanoic acid is 1.8fold higher than wild-type activity, activity with 2-oxo-5-phenylpentanoic acid is identical to wild-type activity. The ratio of turnover number to KM-value for 2-oxo-4-methylpentanoic acid is 10fold higher than the wild-type value, the ratio of turnover number to KM-value for 2-oxo-4-methylhexanoic acid is 1.13fold higher than the wild-type value, the ratio of turnover number to KM-value for benzoylformate is 5.4fold lower than the wild-type value, the ratio of turnover number to KM-value for 2-oxohexanoic acid is 14.1fold higher than the wild-type value, the ratio of turnover number to KM-value for 2-oxopentanoic acid is 4.6fold higher than the wild-type value. Mutation has no effect on the range of products obtained by carboligation of acetaldehyde and benzaldehyde, the yield of the product 2-hydroxypropiophenone an decreases about 3fold and the enantioselectivity of acetoin and 2-hydroxypropiophenone is altered | Pseudomonas putida |
A460I/F464I | activity with pyruvate is higher than wild-type activity, activity with 2-oxobutanoic acid is 1.8fold lower than wild-type activity, activity with 2-oxopentanoic acid is 9.2fold lower than wild-type value, activity with 2-oxohexanoic acid is 3.3fold lower than wild-type activity, activity with 3-methyl-2-oxobutanoic acid is 1.5fold lower than wild-type activity, activity with 3-methyl-2-oxopentanoic acid is 1.2fold lower than wild-type activity, activity with 2-oxo-4-methylpentanoic acid is 1.5fold lower than wild-type activity, activity with 4,4-dimethyl-2-oxopentanoic acid is 10fold lower than wild-type activity, activity with 2-oxo-4-methylhexanoic acid is 1.1fold lower than wild-type activity, activity with benzoylformate is 64.3fold lower than wild-type activity, no activity with 2-oxo-3-phenylpropanoic acid, activity with 2-oxo-4-phenylbutanoic acid is 1.3fold lower than wild-type activity, activity with 2-oxo-5-phenylpentanoic acid is fold higher than wild-type activity. the ratio of turnover number to KM-value for benzoylformate is 88.7fold lower than the wild-type value | Pseudomonas putida |
G464I | activity with pyruvate is higher than wild-type activity, activity with 2-oxobutanoic acid is 1.2fold lower than wild-type activity, activity with 2-oxopentanoic acid is 4.8fold lower than wild-type value, activity with 2-oxohexanoic acid is 1.6fold lower than wild-type activity, activity with 3-methyl-2-oxobutanoic acid is 1.1fold higher than wild-type activity, activity with 3-methyl-2-oxopentanoic acid is 1.3fold higher than wild-type activity, activity with 2-oxo-4-methylpentanoic acid is 1.1fold higher than wild-type activity, activity with 4,4-dimethyl-2-oxopentanoic acid is identical to wild-type activity, activity with 2-oxo-4-methylhexanoic acid is 1.3fold higher than wild-type activity, activity with, activity with benzoylformate is 9.2fold lower than wild-type activity, activity with 2-oxo-3-phenylpropanoic acid is identical to wild-type activity, activity with 2-oxo-4-phenylbutanoic acid is9.3fold lower than wild-type activity, no activity with 2-oxo-5-phenylpentanoic acid. The ratio of turnover number to KM-value for benzoylformate is 31.8fold lower than the wild-type value. Mutation has no effect on the range of products obtained by carboligation of acetaldehyde and benzaldehyde, the yield of the product 2-hydroxypropiophenone an decreases about 3fold and the enantioselectivity of acetoin and 2-hydroxypropiophenone is altered | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
2-oxo-4-methylhexanoic acid | substrate inhibition | Pseudomonas putida | |
2-oxohexanoic acid | substrate inhibition | Pseudomonas putida | |
2-oxopentanoic acid | substrate inhibition | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | KM-value of wild-type enzyme for 2-oxo-4-methylhexanoic acid (pH 6.0, 30°C) is above 20 mM | Pseudomonas putida | |
0.2 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
0.8 | - |
benzoylformate | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
1 | - |
2-oxohexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
1.1 | - |
2-oxo-4-methylhexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
1.1 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme A460I/F464I | Pseudomonas putida | |
1.2 | - |
2-oxopentanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
2.3 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme F464I | Pseudomonas putida | |
3.4 | - |
2-oxo-4-methylpentanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
4.1 | - |
2-oxohexanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
6 | - |
2-oxopentanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
9.6 | - |
2-oxo-4-methylpentanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
18.3 | - |
2-oxobutanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | P20906 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-oxo-3-phenylpropanoic acid | - |
Pseudomonas putida | phenylacetaldehyde + CO2 | - |
? | |
2-oxo-4-methylhexanoic acid | - |
Pseudomonas putida | 3-methylpentanal + CO2 | - |
? | |
2-oxo-4-methylpentanoic acid | - |
Pseudomonas putida | 3-methylbutanal + CO2 | - |
? | |
2-oxo-4-phenylbutanoic acid | - |
Pseudomonas putida | 3-phenylpropionaldehyde + CO2 | - |
? | |
2-oxo-5-phenylpentanoic acid | - |
Pseudomonas putida | 4-phenylpentanal + CO2 | - |
? | |
2-oxobutanoic acid | - |
Pseudomonas putida | propionaldehyde + CO2 | - |
? | |
2-oxohexanoic acid | - |
Pseudomonas putida | valeraldehyde + CO2 | - |
? | |
2-oxopentanoic acid | - |
Pseudomonas putida | butyraldehyde + CO2 | - |
? | |
3 benzaldehyde + acetaldehyde | - |
Pseudomonas putida | (R)-benzoin + (S)-2-hydroxypropiophenone | - |
? | |
3-methyl-2-oxobutanoic acid | - |
Pseudomonas putida | 2-methylpropanal + CO2 | - |
? | |
3-methyl-2-oxopentanoic acid | - |
Pseudomonas putida | 2-methylbutanal + CO2 | - |
? | |
4,4-dimethyl-2-oxopentanoic acid | - |
Pseudomonas putida | 3,3-dimethylbutanal + CO2 | - |
? | |
benzoylformate | best substrate for wild-type enzyme and mutant enzymes A460I, F464I, and A4670I, F464I | Pseudomonas putida | benzaldehyde + CO2 | - |
? | |
additional information | no activity with 3,3-dimethyl-2-oxopentanoic acid and cyclohexaneglyoxylic acid (wild-type enzyme and mutant enzymes A460I, F464I and A460I/F464I) | Pseudomonas putida | ? | - |
? | |
pyruvate | activity of wild-type enzyme is very low, very low activity with mutant enzymes A460I, F464I, and A4670I, F464I | Pseudomonas putida | acetaldehyde + CO2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
tetramer | - |
Pseudomonas putida |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
8.6 | - |
2-oxo-4-methylhexanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
9.8 | - |
2-oxo-4-methylhexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
11 | - |
2-oxo-4-methylpentanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
17.3 | - |
2-oxobutanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
18.8 | - |
2-oxobutanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
21 | - |
2-oxohexanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
21 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme A460I/F464I | Pseudomonas putida | |
38 | - |
2-oxo-4-methylpentanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
40.6 | - |
2-oxopentanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
44.3 | - |
2-oxopentanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
62 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
71.8 | - |
2-oxohexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
123 | - |
benzoylformate | pH 6.0, 30°C, mutant enzyme F464I | Pseudomonas putida | |
1349 | - |
benzoylformate | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
thiamine diphosphate | dependent on | Pseudomonas putida |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | 5 | 2-oxohexanoic acid | pH 6.0, 30°C, wild-type enzyme | Pseudomonas putida | |
10 | - |
2-oxohexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida | |
20 | - |
2-oxopentanoic acid | pH 6.0, 30°C, wild-type enzyme and mutant enzyme A460I | Pseudomonas putida | |
48 | - |
2-oxo-4-methylhexanoic acid | pH 6.0, 30°C, mutant enzyme A460I | Pseudomonas putida |