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Literature summary 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)

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

Protein Variants

EC Number Protein Variants Comment Organism
4.1.1.1 I472A/I476F increase in substrate binding affinity and specificity, highest enantioselectivity for (S)-acetoin, very low yield of product Zymomonas mobilis
4.1.1.1 I476F rapid loss of cofactor thiamine diphosphate. Improvement of enantioselectivity for (S)-acetoin Zymomonas mobilis
4.1.1.1 mutant I472A 2fold decrease in pyruvate decarboxylase activity, switch in substrate specificity to catalyse decarboxylation of benzoylformate, chimera between pyruvate decarboxylase and benzoylformate decarboxylase. Preferred substrates are 2-ketopentanoic acid and 2-ketohexanoic acid. Improvement of enantioselectivity for (S)-acetoin Zymomonas mobilis
4.1.1.7 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
4.1.1.7 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
4.1.1.7 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

EC Number Inhibitors Comment Organism Structure
4.1.1.7 2-oxo-4-methylhexanoic acid substrate inhibition Pseudomonas putida
4.1.1.7 2-oxohexanoic acid substrate inhibition Pseudomonas putida
4.1.1.7 2-oxopentanoic acid substrate inhibition Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.1.1 0.2
-
2-ketohexanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 0.5
-
2-ketohexanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 1.1
-
pyruvate wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 1.7
-
2-keto-4-methylhexanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 1.8
-
benzoylformate mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 2.5
-
2-ketopentanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 2.6
-
pyruvate mutant I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 3.7
-
2-keto-4-methylhexanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 4.4
-
benzoylformate mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 4.7
-
2-ketobutanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 6.7
-
2-ketobutanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 7.6
-
2-ketopentanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 7.8
-
pyruvate mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 11
-
2-ketopentanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 12.7
-
2-ketohexanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 40
-
2-keto-4-methylhexanoic acid and above, wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 50
-
pyruvate mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 50
-
2-ketobutanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.7 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
4.1.1.7 0.2
-
benzoylformate pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 0.8
-
benzoylformate pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 1
-
2-oxohexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 1.1
-
2-oxo-4-methylhexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 1.1
-
benzoylformate pH 6.0, 30°C, mutant enzyme A460I/F464I Pseudomonas putida
4.1.1.7 1.2
-
2-oxopentanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 2.3
-
benzoylformate pH 6.0, 30°C, mutant enzyme F464I Pseudomonas putida
4.1.1.7 3.4
-
2-oxo-4-methylpentanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 4.1
-
2-oxohexanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 6
-
2-oxopentanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 9.6
-
2-oxo-4-methylpentanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 18.3
-
2-oxobutanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.1.1.1 Mg2+ required Zymomonas mobilis

Organism

EC Number Organism UniProt Comment Textmining
4.1.1.1 Zymomonas mobilis P06672
-
-
4.1.1.7 Pseudomonas putida P20906
-
-

Purification (Commentary)

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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.1.1 2-keto-4-methylhexanoic acid
-
Zymomonas mobilis 3-methylpentanal + CO2
-
?
4.1.1.1 2-ketobutanoic acid
-
Zymomonas mobilis propanal + CO2
-
?
4.1.1.1 2-ketohexanoic acid
-
Zymomonas mobilis pentanal + CO2
-
?
4.1.1.1 2-ketopentanoic acid
-
Zymomonas mobilis butanal + CO2
-
?
4.1.1.1 benzoylformate substrate only for mutant mutant I472A Zymomonas mobilis benzaldehyde + CO2
-
?
4.1.1.1 pyruvate
-
Zymomonas mobilis acetaldehyde + CO2
-
?
4.1.1.7 2-oxo-3-phenylpropanoic acid
-
Pseudomonas putida phenylacetaldehyde + CO2
-
?
4.1.1.7 2-oxo-4-methylhexanoic acid
-
Pseudomonas putida 3-methylpentanal + CO2
-
?
4.1.1.7 2-oxo-4-methylpentanoic acid
-
Pseudomonas putida 3-methylbutanal + CO2
-
?
4.1.1.7 2-oxo-4-phenylbutanoic acid
-
Pseudomonas putida 3-phenylpropionaldehyde + CO2
-
?
4.1.1.7 2-oxo-5-phenylpentanoic acid
-
Pseudomonas putida 4-phenylpentanal + CO2
-
?
4.1.1.7 2-oxobutanoic acid
-
Pseudomonas putida propionaldehyde + CO2
-
?
4.1.1.7 2-oxohexanoic acid
-
Pseudomonas putida valeraldehyde + CO2
-
?
4.1.1.7 2-oxopentanoic acid
-
Pseudomonas putida butyraldehyde + CO2
-
?
4.1.1.7 3 benzaldehyde + acetaldehyde
-
Pseudomonas putida (R)-benzoin + (S)-2-hydroxypropiophenone
-
?
4.1.1.7 3-methyl-2-oxobutanoic acid
-
Pseudomonas putida 2-methylpropanal + CO2
-
?
4.1.1.7 3-methyl-2-oxopentanoic acid
-
Pseudomonas putida 2-methylbutanal + CO2
-
?
4.1.1.7 4,4-dimethyl-2-oxopentanoic acid
-
Pseudomonas putida 3,3-dimethylbutanal + CO2
-
?
4.1.1.7 benzoylformate best substrate for wild-type enzyme and mutant enzymes A460I, F464I, and A4670I, F464I Pseudomonas putida benzaldehyde + CO2
-
?
4.1.1.7 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 ?
-
?
4.1.1.7 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

EC Number Subunits Comment Organism
4.1.1.7 tetramer
-
Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.1.1.1 0.4
-
2-keto-4-methylhexanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 0.8
-
2-ketohexanoic acid mutant I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 1.2
-
benzoylformate mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 4
-
2-ketohexanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 6.9
-
benzoylformate mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 7.7
-
2-keto-4-methylhexanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 8
-
pyruvate mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 8.1
-
2-keto-4-methylhexanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 9
-
2-ketobutanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 11
-
2-ketopentanoic acid mutant I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 30
-
2-ketopentanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 32
-
2-ketobutanoic acid mutant I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 53
-
2-ketopentanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 77
-
pyruvate mutant I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 82
-
2-ketohexanoic acid mutant I472A/I476F, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 130
-
2-ketohexanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 200
-
pyruvate mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 220
-
2-ketopentanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 250
-
2-ketobutanoic acid mutant I472A, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 320
-
2-ketobutanoic acid wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.1 486
-
pyruvate wild type, pH 6.5, 30°C Zymomonas mobilis
4.1.1.7 8.6
-
2-oxo-4-methylhexanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 9.8
-
2-oxo-4-methylhexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 11
-
2-oxo-4-methylpentanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 17.3
-
2-oxobutanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 18.8
-
2-oxobutanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 21
-
2-oxohexanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 21
-
benzoylformate pH 6.0, 30°C, mutant enzyme A460I/F464I Pseudomonas putida
4.1.1.7 38
-
2-oxo-4-methylpentanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 40.6
-
2-oxopentanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 44.3
-
2-oxopentanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 62
-
benzoylformate pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 71.8
-
2-oxohexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 123
-
benzoylformate pH 6.0, 30°C, mutant enzyme F464I Pseudomonas putida
4.1.1.7 1349
-
benzoylformate pH 6.0, 30°C, wild-type enzyme Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
4.1.1.1 thiamine diphosphate
-
Zymomonas mobilis
4.1.1.7 thiamine diphosphate dependent on Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.1.1.7 2 5 2-oxohexanoic acid pH 6.0, 30°C, wild-type enzyme Pseudomonas putida
4.1.1.7 10
-
2-oxohexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida
4.1.1.7 20
-
2-oxopentanoic acid pH 6.0, 30°C, wild-type enzyme and mutant enzyme A460I Pseudomonas putida
4.1.1.7 48
-
2-oxo-4-methylhexanoic acid pH 6.0, 30°C, mutant enzyme A460I Pseudomonas putida