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Literature summary for 4.1.1.7 extracted from

  • Siegert, P.; McLeish, M.J.; Baumann, M.; Iding, H.; Kneen, M.M.; Kenyon, G.L.; Pohl, M.
    Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida (2005), Protein Eng. Des. Sel., 18, 345-357.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
wild-type and mutant enzymes A460I, F464I and A460I/F464I Pseudomonas putida

Protein Variants

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

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 [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

Organism UniProt Comment Textmining
Pseudomonas putida P20906
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes Pseudomonas putida

Substrates and Products (Substrate)

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

Subunits Comment Organism
tetramer
-
Pseudomonas putida

Turnover Number [1/s]

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

Cofactor Comment Organism Structure
thiamine diphosphate dependent on Pseudomonas putida

Ki Value [mM]

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