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Literature summary for 1.13.11.15 extracted from
- 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa an Fe(II)-containing enzyme with fast turnover (2017), PLoS ONE, 12, e0171135 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene dhpao, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression in Escherichia coli strain BL21 (DE3) | Pseudomonas aeruginosa |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| DTT | destabilizes the enzyme | Pseudomonas aeruginosa |  |
| EDTA | destabilizes the enzyme | Pseudomonas aeruginosa | |
| Fe3+ | striong inhibition | Pseudomonas aeruginosa | |
| H2O2 | destabilizes the enzyme | Pseudomonas aeruginosa | |
| Mn2+ | slight inhibition | Pseudomonas aeruginosa | |
| additional information | NaF no effect on the enzyme stability and activity, no significant inhibition by Mg2+ | Pseudomonas aeruginosa | |
| Na2SO4 | destabilizes the enzyme | Pseudomonas aeruginosa | |
| NaN3 | slightly destabilizes the enzyme | Pseudomonas aeruginosa | |
| Zn2+ | slight inhibition | Pseudomonas aeruginosa | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 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 | |
| 0.058 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C, in presence of ascorbate | Pseudomonas aeruginosa | |
| 0.067 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C | Pseudomonas aeruginosa | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| ascorbate | activates at 0.5 mM | Pseudomonas aeruginosa | |
| Fe(NH4)2(SO4)2 | activates at 0.5 mM, activation is enhanced by 10 mM DTT | Pseudomonas aeruginosa | |
| Fe2+ | 0.71 mol/subunit, dependent on, a mononuclear non-heme ferrous ion, Fe(II), is the metal cofactor | Pseudomonas aeruginosa | |
| 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]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 112200 | - |
recombinant enzyme, gel filtration | Pseudomonas aeruginosa |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 3,4-dihydroxyphenylacetate + O2 | Pseudomonas aeruginosa | - |
2-hydroxy-5-carboxymethylmuconate semialdehyde | - |
? | |
| 3,4-dihydroxyphenylacetate + O2 | Pseudomonas aeruginosa PAO | - |
2-hydroxy-5-carboxymethylmuconate semialdehyde | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Pseudomonas aeruginosa | - |
- |
- |
| Pseudomonas aeruginosa PAO | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 44.3 | - |
purified recombinant enzyme, pH 7.5, 25°C | Pseudomonas aeruginosa |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 3,4-dihydroxyphenylacetate + O2 | - |
Pseudomonas aeruginosa | 2-hydroxy-5-carboxymethylmuconate semialdehyde | - |
? | |
| 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 | - |
? | |
| 3,4-dihydroxyphenylacetate + O2 | - |
Pseudomonas aeruginosa PAO | 2-hydroxy-5-carboxymethylmuconate semialdehyde | - |
? | |
| 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 | - |
? | |
| 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 | ? | - |
? | |
| 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
| Subunits | Comment | Organism |
|---|---|---|
| homotetramer | 4 * 30000, recombinant enzyme, SDS-PAGE | Pseudomonas aeruginosa |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| DHPAO | - |
Pseudomonas aeruginosa |
| homoprotocatechuate 2,3 dioxygenase | - |
Pseudomonas aeruginosa |
| HPCD | - |
Pseudomonas aeruginosa |
| PaDHPAO | - |
Pseudomonas aeruginosa |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 55 | - |
- |
Pseudomonas aeruginosa |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 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]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 62.4 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C | Pseudomonas aeruginosa | |
| 63.5 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C, in presence of ascorbate | Pseudomonas aeruginosa | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
- |
Pseudomonas aeruginosa |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 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
| General Information | Comment | Organism |
|---|---|---|
| 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]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 900 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C | Pseudomonas aeruginosa | |
| 1100 | - |
3,4-Dihydroxyphenylacetate | pH 7.5, 25°C, in presence of ascorbate | Pseudomonas aeruginosa | |
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