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Literature summary extracted from

  • Pornsuwan, S.; Maenpuen, S.; Kamutira, P.; Watthaisong, P.; Thotsaporn, K.; Tongsook, C.; Juttulapa, M.; Nijvipakul, S.; Chaiyen, P.
    3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa an Fe(II)-containing enzyme with fast turnover (2017), PLoS ONE, 12, e0171135 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.15 gene dhpao, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression in Escherichia coli strain BL21 (DE3) Pseudomonas aeruginosa

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.13.11.15 DTT destabilizes the enzyme Pseudomonas aeruginosa
1.13.11.15 EDTA destabilizes the enzyme Pseudomonas aeruginosa
1.13.11.15 Fe3+ striong inhibition Pseudomonas aeruginosa
1.13.11.15 H2O2 destabilizes the enzyme Pseudomonas aeruginosa
1.13.11.15 Mn2+ slight inhibition Pseudomonas aeruginosa
1.13.11.15 additional information NaF no effect on the enzyme stability and activity, no significant inhibition by Mg2+ Pseudomonas aeruginosa
1.13.11.15 Na2SO4 destabilizes the enzyme Pseudomonas aeruginosa
1.13.11.15 NaN3 slightly destabilizes the enzyme Pseudomonas aeruginosa
1.13.11.15 Zn2+ slight inhibition Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.15 additional information
-
 additional information Michaelis-Menten steady-state kinetics under air-saturated conditions. The rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different Pseudomonas aeruginosa
1.13.11.15 0.058
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C, in presence of ascorbate Pseudomonas aeruginosa
1.13.11.15 0.067
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C Pseudomonas aeruginosa

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.15 ascorbate activates at 0.5 mM Pseudomonas aeruginosa
1.13.11.15 Fe(NH4)2(SO4)2 activates at 0.5 mM, activation is enhanced by 10 mM DTT Pseudomonas aeruginosa
1.13.11.15 Fe2+ 0.71 mol/subunit, dependent on, a mononuclear non-heme ferrous ion, Fe(II), is the metal cofactor Pseudomonas aeruginosa
1.13.11.15 additional information apoPaDHPAO cannot be restored by substitution with either Mn(II) or Co(II), although the two metal ions can bind to apoPaDHPAO, Fe(II) is the native and mandatory metal ion for PaDHPAO. 10 mM DTT alone does not activate the enzyme Pseudomonas aeruginosa

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.13.11.15 112200
-
 recombinant enzyme, gel filtration Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.15 3,4-dihydroxyphenylacetate + O2 Pseudomonas aeruginosa
-
 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?
1.13.11.15 3,4-dihydroxyphenylacetate + O2 Pseudomonas aeruginosa PAO
-
 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.15 Pseudomonas aeruginosa
-
-
-
1.13.11.15 Pseudomonas aeruginosa PAO
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.15 recombinant soluble enzyme 2.5fold from Escherichia coli strain BL21 (DE3) by anion exchange chromatography, ultrafiltration, hydrophobic interaction chromatography, ultrafiltration, and gel filtration Pseudomonas aeruginosa

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.13.11.15 44.3
-
 purified recombinant enzyme, pH 7.5, 25°C Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.15 3,4-dihydroxyphenylacetate + O2
-
 Pseudomonas aeruginosa 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?
1.13.11.15 3,4-dihydroxyphenylacetate + O2 the enzyme catalyzes the extradiol ring cleavage of 3,4-dihydroxyphenylacetate (DHPA) by incorporation of molecular oxygen to yield 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS). O2 insertion occurs through the substrate-alkylperoxo-Fe(II) intermediate Pseudomonas aeruginosa 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?
1.13.11.15 3,4-dihydroxyphenylacetate + O2
-
 Pseudomonas aeruginosa PAO 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?
1.13.11.15 3,4-dihydroxyphenylacetate + O2 the enzyme catalyzes the extradiol ring cleavage of 3,4-dihydroxyphenylacetate (DHPA) by incorporation of molecular oxygen to yield 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS). O2 insertion occurs through the substrate-alkylperoxo-Fe(II) intermediate Pseudomonas aeruginosa PAO 2-hydroxy-5-carboxymethylmuconate semialdehyde
-
 ?
1.13.11.15 additional information poor or no activity with 3,4-dihydroxyphenylpropionate, 3,4-dihydroxybenzoate, 4-hydroxyphenylacetate, 4-hydroxybenzoate, 4-hydroxyphenylpropionate, 4-hydroxy-3-methoxyphenylacetate, and 4-nitrocatechol Pseudomonas aeruginosa ?
-
 ?
1.13.11.15 additional information poor or no activity with 3,4-dihydroxyphenylpropionate, 3,4-dihydroxybenzoate, 4-hydroxyphenylacetate, 4-hydroxybenzoate, 4-hydroxyphenylpropionate, 4-hydroxy-3-methoxyphenylacetate, and 4-nitrocatechol Pseudomonas aeruginosa PAO ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.15 homotetramer 4 * 30000, recombinant enzyme, SDS-PAGE Pseudomonas aeruginosa

Synonyms

EC Number Synonyms Comment Organism
1.13.11.15 DHPAO
-
 Pseudomonas aeruginosa
1.13.11.15 homoprotocatechuate 2,3 dioxygenase
-
 Pseudomonas aeruginosa
1.13.11.15 HPCD
-
 Pseudomonas aeruginosa
1.13.11.15 PaDHPAO
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.15 55
-
-
 Pseudomonas aeruginosa

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.13.11.15 5 70 activity range, profile overview. The enzyme activity increases as temperature increases up to 55°C and then decreases at higher temperatures. The rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different Pseudomonas aeruginosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.15 62.4
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C Pseudomonas aeruginosa
1.13.11.15 63.5
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C, in presence of ascorbate Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.15 7.5
-
-
 Pseudomonas aeruginosa

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.13.11.15 5.5 9.5 activity range, profile overview. pH-rate profile of enzyme PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of pH 6.5 and pH 8.9 Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
1.13.11.15 evolution the enzyme PaDHPAO likely belongs to the mononuclear non-heme metal-containing 2-His-1-carboxylate facial triad enzyme superfamily Pseudomonas aeruginosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.15 900
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C Pseudomonas aeruginosa
1.13.11.15 1100
-
 3,4-Dihydroxyphenylacetate pH 7.5, 25°C, in presence of ascorbate Pseudomonas aeruginosa