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Literature summary extracted from

  • Lingen, B.; Grotzinger, J.; Kolter, D.; Kula, M.R.; Pohl, M.
    Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis (2002), Protein Eng., 15, 585-593.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.1.7 expressed in Escherichia coli strain SG13009/pREP4 Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
4.1.1.7 L476A has a 4.11fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476C has a 4.31fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476G has a 4.27fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476H has a 3.19fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476K has a 5fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476M has a 4.23fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476P decreased activity Pseudomonas putida
4.1.1.7 L476Q has a 5.06fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476Q/S535G decreased activity Pseudomonas putida
4.1.1.7 L476S has a 3.5fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 L476T has a 3.98fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 S181T has a 1.75fold higher carboligase activity than the wild type enzyme Pseudomonas putida
4.1.1.7 S181T/L476P increased activity Pseudomonas putida

General Stability

EC Number General Stability Organism
4.1.1.7 the wild type enzyme shows no loss of activity in cofactor-free buffer over 24 h Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.1.1.7 benzoylformate
-
 Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.1.7 0.54
-
 benzoylformate wild type enzyme, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 0.58
-
 benzoylformate mutant enzyme L476Q, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 0.59
-
 benzoylformate mutant enzyme L476G, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 0.6
-
 benzoylformate mutant enzyme S181T/L476P, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1
-
 benzoylformate mutant enzyme L476H, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.02
-
 benzoylformate mutant enzyme L476T, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.03
-
 benzoylformate mutant enzyme L476P, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.06
-
 benzoylformate mutant enzyme L476Q/S525G, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.08
-
 benzoylformate mutant enzyme L476M, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.14
-
 benzoylformate mutant enzyme L476S, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.17
-
 benzoylformate mutant enzyme L476A, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.23
-
 benzoylformate mutant enzyme S181T, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.26
-
 benzoylformate mutant enzyme L476C, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida
4.1.1.7 1.46
-
 benzoylformate mutant enzyme L476K, in 50 mM potassium phosphate buffer pH 7.0, 0.5 mM thiamine diphosphate, 2.5 mM MgSO4, at 30°C Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.1.1.7 Mg2+ required Pseudomonas putida

Organic Solvent Stability

EC Number Organic Solvent Comment Organism
4.1.1.7 DMSO retains its full activity after 10 days of incubation in DMSO (20% v/v) at 25°C Pseudomonas putida
4.1.1.7 Ethanol retains its full activity after 10 days of incubation in aqueous ethanol (1.5 M) at 25°C Pseudomonas putida

Organism

EC Number Organism UniProt Comment Textmining
4.1.1.7 Pseudomonas putida
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.1.1.7 Ni-NTA column chromatography Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.1.7 benzaldehyde + acetaldehyde
-
 Pseudomonas putida (S)-2-hydroxy-1-phenyl-propanone
-
 ?
4.1.1.7 benzoylformate
-
 Pseudomonas putida benzaldehyde + CO2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.1.1.7 BFD
-
 Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.1.1.7 8
-
 the optimal pH is 8.0 or above for the carboligase reaction Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
4.1.1.7 thiamine diphosphate dependent Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.1.1.7 45
-
 benzoylformate mutant enzyme L476P Pseudomonas putida
4.1.1.7 143
-
 benzoylformate wild type enzyme Pseudomonas putida