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Literature summary for 1.14.14.20 extracted from
- Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD (2003), J. Biol. Chem., 278, 47545-47553.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| flavin reductase PheA2 | the two-protein system phenol hydroxylase consists of an oxygenase (PheA1) and a flavin reductase (PheA2). PheA1 shows almost no phenol hydroxylase activity when assayed at 50°C and pH 7.0 in absence of PheA2. The PheA1-mediated conversion of phenol to catechol is strongly stimulated in presence of catalytic amounts of PheA2 | Parageobacillus thermoglucosidasius | |
| phenol hydroxylase PheA2 | PheA1-mediated conversion of phenol to catechol is strongly stimulated in the presence of catalytic amounts of PheA2 | Parageobacillus thermoglucosidasius |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene pheA1, recombinant expression in Escherichia coli, tandem expression with pheA2 does not result in PheA2 protein | Parageobacillus thermoglucosidasius |
| gene pheA2, recombinant expression in Escherichia coli, tandem expression with pheA1 does not result in PheA2 protein | Parageobacillus thermoglucosidasius |
| PheA1 and PheA2 are separately expressed in recombinant Escherichia coli BL21 pLysS cells | Parageobacillus thermoglucosidasius |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetic analysis of PheA2, NADH-dependent activities of PheA2 with different electron acceptors, overview | Parageobacillus thermoglucosidasius | |
| 0.0016 | - |
FAD | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius |  |
| 0.0055 | - |
FMN | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
| 0.0055 | - |
riboflavin | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| additional information | - |
PheA1 has a MW of 120000 Da as determined by gel filtration. PheA2 has a MW of 35000 Da as determined by gel filtration | Parageobacillus thermoglucosidasius |
| 18000 | - |
the oxygenase component PheA1 is a dimer: 2 * 57000, SDS-PAGE. The flavin reductase PheA2 is a dimer: 2 * 18000, SDS-PAGE | Parageobacillus thermoglucosidasius |
| 18000 | - |
2 * 18000, about, recombinant PheA2 protein, SDS-PAGE | Parageobacillus thermoglucosidasius |
| 35000 | - |
recombinant PheA2 protein, gel filtration | Parageobacillus thermoglucosidasius |
| 57000 | - |
the oxygenase component PheA1 is a dimer: 2 * 57000, SDS-PAGE. The flavin reductase PheA2 is a dimer: 2 * 18000, SDS-PAGE | Parageobacillus thermoglucosidasius |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | Parageobacillus thermoglucosidasius | enzyme components PheA2 and PheA1 show no protein-protein interaction | ? | - |
? | |
| additional information | Parageobacillus thermoglucosidasius A7 | enzyme components PheA2 and PheA1 show no protein-protein interaction | ? | - |
? | |
| phenol + FADH2 + O2 | Parageobacillus thermoglucosidasius | - |
catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | Parageobacillus thermoglucosidasius A7 | - |
catechol + FAD + H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Parageobacillus thermoglucosidasius | - |
- |
- |
| Parageobacillus thermoglucosidasius | Q9LAG2 | PheA2; or Bacillus thermoglucosidasius, gene pheA2 | - |
| Parageobacillus thermoglucosidasius | Q9LAG3 | PheA1; or Bacillus thermoglucosidasius, gene pheA1 | - |
| Parageobacillus thermoglucosidasius A7 | - |
- |
- |
| Parageobacillus thermoglucosidasius A7 | Q9LAG2 | PheA2; or Bacillus thermoglucosidasius, gene pheA2 | - |
| Parageobacillus thermoglucosidasius A7 | Q9LAG3 | PheA1; or Bacillus thermoglucosidasius, gene pheA1 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| PheA1 and PheA2 expressed in recombinant Escherichia coli BL21 pLysS cells | Parageobacillus thermoglucosidasius |
| recombinant PheA1 from Escherichia coli by anion exchange and hydroxyapatite chromatography, followed by gel filtration | Parageobacillus thermoglucosidasius |
| recombinant PheA2 from Escherichia coli by anion exchange and hydroxyapatite chromatography, followed by gel filtration | Parageobacillus thermoglucosidasius |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 0.004 | - |
specific NADH:FAD reductase activity, pH 7.0 50°C, in absence of phenol hydroxylase PheA2 | Parageobacillus thermoglucosidasius |
| 0.32 | - |
specific activity of PheA1 in presence of PheA2 | Parageobacillus thermoglucosidasius |
| 0.32 | - |
specific NADH:FAD reductase activity, pH 7.0 50°C, in presence of 1 mM phenol hydroxylase PheA2 | Parageobacillus thermoglucosidasius |
| 802 | - |
specific NADH:FAD reductase activity of purified recombinant PheA2, pH 7.0, 53°C | Parageobacillus thermoglucosidasius |
Storage Stability
| Storage Stability | Organism |
|---|---|
| -70°C, no significant loss of activity of the flavin reductase component PheA2 after 4 months | Parageobacillus thermoglucosidasius |
| -70°C, purified recombinant PheA1 protein is not very stable when stored at -70°C because it forms aggregates after thawing | Parageobacillus thermoglucosidasius |
| -70°C, purified recombinant PheA2 protein is stable for 4 months when stored at -70°C | Parageobacillus thermoglucosidasius |
| 4°C, purified recombinant PheA1 is stable when stored as a protein precipitate in 80% ammonium sulfate at 4°C | Parageobacillus thermoglucosidasius |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | enzyme components PheA2 and PheA1 show no protein-protein interaction | Parageobacillus thermoglucosidasius | ? | - |
? | |
| additional information | PheA1 is required for activity of flavin reductase PheA2, no activity by PheA1 or PheA2 alone | Parageobacillus thermoglucosidasius | ? | - |
? | |
| additional information | enzyme components PheA2 and PheA1 show no protein-protein interaction | Parageobacillus thermoglucosidasius A7 | ? | - |
? | |
| additional information | PheA1 is required for activity of flavin reductase PheA2, no activity by PheA1 or PheA2 alone | Parageobacillus thermoglucosidasius A7 | ? | - |
? | |
| phenol + FADH2 + O2 | - |
Parageobacillus thermoglucosidasius | catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | PheA1 is required for activity, no activity by PheA2 alone. PheA2 acts according to a ping pong bi bi reaction mechanism in which NADH reduces the FAD cofactor, which in turn transfers electrons to the FAD substrate | Parageobacillus thermoglucosidasius | catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | - |
Parageobacillus thermoglucosidasius A7 | catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | PheA1 is required for activity, no activity by PheA2 alone. PheA2 acts according to a ping pong bi bi reaction mechanism in which NADH reduces the FAD cofactor, which in turn transfers electrons to the FAD substrate | Parageobacillus thermoglucosidasius A7 | catechol + FAD + H2O | - |
? | |
| phenol + FMNH2 + O2 | - |
Parageobacillus thermoglucosidasius | catechol + FMN + H2O | - |
? | |
| phenol + FMNH2 + O2 | - |
Parageobacillus thermoglucosidasius A7 | catechol + FMN + H2O | - |
? | |
| phenol + NADH + H+ + O2 | the two-protein system phenol hydroxylase consists of an oxygenase (PheA1) and a flavin reductase (PheA2). PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. PheA1 catalyzes the NADH-dependent reduction of free flavins according to a ping pong bi bi mechanism | Parageobacillus thermoglucosidasius | catechol + NAD+ + H2O | - |
? | |
| phenol + NADH + H+ + O2 | the two-protein system phenol hydroxylase consists of an oxygenase (PheA1) and a flavin reductase (PheA2). PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. PheA1 catalyzes the NADH-dependent reduction of free flavins according to a ping pong bi bi mechanism | Parageobacillus thermoglucosidasius A7 | catechol + NAD+ + H2O | - |
? | |
| phenol + reduced riboflavin + O2 | - |
Parageobacillus thermoglucosidasius | catechol + riboflavin + H2O | - |
? | |
| phenol + reduced riboflavin + O2 | - |
Parageobacillus thermoglucosidasius A7 | catechol + riboflavin + H2O | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | 2 * 18000, about, recombinant PheA2 protein, SDS-PAGE | Parageobacillus thermoglucosidasius |
| More | the oxygenase component PheA1 is a dimer: 2 * 57000, SDS-PAGE. The flavin reductase PheA2 is a dimer: 2 * 18000, SDS-PAGE | Parageobacillus thermoglucosidasius |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PheA | the two-protein system phenol hydroxylase, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) | Parageobacillus thermoglucosidasius |
| phenol hydroxylase | the two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) | Parageobacillus thermoglucosidasius |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 50 | - |
assay at | Parageobacillus thermoglucosidasius |
| 55 | - |
phenol hydroxylase system | Parageobacillus thermoglucosidasius |
| 55 | - |
NADH:FAD reductase activity of PheA2 | Parageobacillus thermoglucosidasius |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | 65 | activity range, profile overview | Parageobacillus thermoglucosidasius |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 60 | - |
PheA1, stable for 2 h | Parageobacillus thermoglucosidasius |
| 60 | - |
purified recombinant PheA1, completely stable for 2 h | Parageobacillus thermoglucosidasius |
| 65 | - |
2 h, more than 65% of the NADH:FAD reductase activity of PheA2 is maintained | Parageobacillus thermoglucosidasius |
| 65 | - |
purified recombinant PheA2, 2 h, 65% NADH:FAD reductase activity remaining | Parageobacillus thermoglucosidasius |
| 70 | - |
complete inactivation of Phea1 after 10 min | Parageobacillus thermoglucosidasius |
| 70 | - |
purified recombinant PheA1, complete inactivation within 10 min | Parageobacillus thermoglucosidasius |
| 85 | - |
purified recombinant PheA2, 2 h, 15-20% NADH:FAD reductase activity remaining | Parageobacillus thermoglucosidasius |
| 88 | - |
2 h, 15-20% of the NADH:FAD reductase activity of PheA2 is maintained | Parageobacillus thermoglucosidasius |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 43 | - |
FAD | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
| 147 | - |
riboflavin | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
| 180 | - |
FMN | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| additional information | - |
the pH-optimum of the NADH:FAD reductase activity of PheA2 is pH 6.7 | Parageobacillus thermoglucosidasius |
| 6.7 | - |
NADH:FAD reductase activity of PheA2 | Parageobacillus thermoglucosidasius |
| 7 | - |
assay at | Parageobacillus thermoglucosidasius |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 4 | 9 | activity range, profile overview | Parageobacillus thermoglucosidasius |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | PheA2 uses FAD both as a substrate and as a prosthetic group, strictly dependent on, neither FMN nor riboflavin can replace FAD in this reaction. PheA2 is a a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group | Parageobacillus thermoglucosidasius | |
| FADH2 | FAD is bound to PheA2, binding structure analysis, overview | Parageobacillus thermoglucosidasius | |
| FMN | - |
Parageobacillus thermoglucosidasius | |
| additional information | the free FAD acts as a true substrate. In addition to FAD, PheA2 is also active with FMN and riboflavin. The turnover rate of PheA2 with free flavins is strongly dependent on temperature. At 25°C, the activity with FMN and riboflavin is much higher than with FAD, but when the temperature is raised to 53°C, the turnover rates with the different flavins becomes nearly identical. Dichlorophenolindophenol is a poor cofactor | Parageobacillus thermoglucosidasius | |
| NADH | - |
Parageobacillus thermoglucosidasius | |
| NADH | NADH is a much better coenzyme for PheA2 than NADPH | Parageobacillus thermoglucosidasius | |
| riboflavin | - |
Parageobacillus thermoglucosidasius | |
pI Value
| Organism | Comment | pI Value Maximum | pI Value |
|---|---|---|---|
| Parageobacillus thermoglucosidasius | isoelectric focusing | - |
5.2 |
| Parageobacillus thermoglucosidasius | PheA1 and PheA2, isoelectric focusing | - |
5.2 |
| Parageobacillus thermoglucosidasius | sequence calculation | - |
5.36 |
| Parageobacillus thermoglucosidasius | sequence calculation | - |
6.29 |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | PheA2 homology modeling using the structure of ferric reductase (FeR) from Archaeoglobus fulgidus with NADP+ bound, PDB ID 1IOS, as template, overview | Parageobacillus thermoglucosidasius |
| physiological function | phenol hydroxylase (PheA) catalyzes the first step in the degradation of phenol in Geobacillus thermoglucosidasius strain A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) | Parageobacillus thermoglucosidasius |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 26700 | - |
riboflavin | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
| 26900 | - |
FAD | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
| 32700 | - |
FMN | NADH-dependent reduction, 25°C, pH 7.0, recombinant PheA2 | Parageobacillus thermoglucosidasius | |
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