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Literature summary extracted from

  • Wang, W.; Mazurkewich, S.; Kimber, M.S.; Seah, S.Y.
    Structural and kinetic characterization of 4-hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolase, a protocatechuate degradation enzyme evolutionarily convergent with the HpaI and DmpG pyruvate aldolases (2010), J. Biol. Chem., 285, 36608-36615.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.3.B3 expressed in Escherichia coli BL21(lambdaDE3) Pseudomonas putida
4.1.3.17 expressed in Escherichia coli BL21(lambdaDE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.1.3.B3 hanging drop method, PEG 4000 Pseudomonas putida
4.1.3.17 hanging drop method, PEG 4000 Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
4.1.3.B3 R123A no activity Pseudomonas putida
4.1.3.B3 R123K retains some activity (substrate 4-hydroxy-4-methyl-2-oxoglutarate) Pseudomonas putida
4.1.3.17 R123A no activity Pseudomonas putida
4.1.3.17 R123K retains some activity Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.1.3.17 oxalate competitive inhibitor Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.1.3.B3 0.03
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.B3 0.066
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.022
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.036
-
 oxaloacetate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C; substrate inhibition constante 0.25 mM Pseudomonas putida
4.1.3.17 0.071
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.15
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.26
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.3
-
 oxaloacetate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C; substrate inhibition constante 0.37 mM Pseudomonas putida
4.1.3.17 5.5
-
 4-hydroxy-4-methyl-2-oxoglutarate R123K mutant protein, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 8.8
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 25
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.1.3.B3 Mg2+
-
 Pseudomonas putida
4.1.3.17 Mg2+ Mn2+: 90.3% activity, Co2+: 64.7% activity, Zn2+: 49.6% activity Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
4.1.3.B3 26000
-
 6 * 26000, crystal structure, gel filtration and SDS-PAGE Pseudomonas putida
4.1.3.17 26000
-
 6 * 26000, crystal structure, gel filtration and SDS-PAGE Pseudomonas putida

Organism

EC Number Organism UniProt Comment Textmining
4.1.3.B3 Pseudomonas putida A5W059
-
-
4.1.3.17 Pseudomonas putida
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.1.3.B3 ion exchange chromatography, hydrophobic interaction chromatography, gel filtration Pseudomonas putida
4.1.3.17 ion exchange chromatography, hydrophobic interaction chromatography, gel filtration Pseudomonas putida

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.1.3.B3 37.7
-
 wild type protein, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 37.7
-
 wild type protein, pH 8.0, 25°C Pseudomonas putida

Storage Stability

EC Number Storage Stability Organism
4.1.3.B3 -80°C, 20 mM sodium HEPES buffer, pH7.5, stable for 2 months Pseudomonas putida
4.1.3.17 -80°C, 20 mM sodium HEPES buffer, pH7.5, stable for 2 months Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.1.3.B3 4-carboxy-4-hydroxy-2-oxoadipate
-
 Pseudomonas putida pyruvate + oxaloacetate
-
 r
4.1.3.17 2-hydroxy-4-oxopentanedioic acid
-
 Pseudomonas putida ?
-
 ?
4.1.3.17 4-hydroxy-2-oxopentanoate
-
 Pseudomonas putida ?
-
 ?
4.1.3.17 4-hydroxy-4-methyl-2-oxoglutarate
-
 Pseudomonas putida 2 pyruvate
-
 r
4.1.3.17 oxaloacetate
-
 Pseudomonas putida ?
-
 ?

Subunits

EC Number Subunits Comment Organism
4.1.3.B3 homohexamer 6 * 26000, crystal structure, gel filtration and SDS-PAGE Pseudomonas putida
4.1.3.17 homohexamer 6 * 26000, crystal structure, gel filtration and SDS-PAGE Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.1.3.B3 7.5
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.B3 14.4
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.048
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.058
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.099
-
 4-hydroxy-4-methyl-2-oxoglutarate R123K mutant protein, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.19
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.25
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 1.52
-
 oxaloacetate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 1.85
-
 oxaloacetate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 13.7
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 19.3
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.1.3.17 additional information
-
 broad optimum (log KCAT) pH 8 - pH 10.5 Pseudomonas putida

pH Range

EC Number pH Minimum pH Maximum Comment Organism
4.1.3.17 6 10.5
-
 Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
4.1.3.17 0.0017
-
 oxalate 1 mM MnCl, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.005
-
 oxalate 1 mM MgCl, pH 8.0, 25°C Pseudomonas putida

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.1.3.B3 220
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.B3 250
-
 4-carboxy-4-hydroxy-2-oxoadipate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.0019
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.022
-
 4-hydroxy-2-oxopentanoate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 0.38
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 3.5
-
 2-hydroxy-4-oxopentanedioic acid wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 6.2
-
 oxaloacetate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 74
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mg2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 420
-
 oxaloacetate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida
4.1.3.17 620
-
 4-hydroxy-4-methyl-2-oxoglutarate wild type protein, 1.0 mM Mn2+, pH 8.0, 25°C Pseudomonas putida