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Literature summary for 1.14.14.20 extracted from
- Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1 (2009), Appl. Microbiol. Biotechnol., 86, 201-211.
Activating Compound
| Activating Compound | Comment | Organism | Structure |
|---|---|---|---|
| additional information | iodoacetamide, phenylmethylsulfonyl fluoride, and EDTA, at 1 mM, do not affect the activity of the pure enzyme | Rhodococcus erythropolis |
Application
| Application | Comment | Organism |
|---|---|---|
| environmental protection | strain UPV-1 is able to grow on phenol as the sole carbon and energy source, removing, concomitantly, the formaldehyde present in phenolic industrial wastewaters | Rhodococcus erythropolis |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| phenol hydroxylase is a two-component flavin-dependent monooxygenase, the two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome, DNA and amino acid sequence determination and analysis, recombinant expression of His6-tagged PheA1 in Escherichia coli strain M15 by nickel affinity chromatography | Rhodococcus erythropolis |
| phenol hydroxylase is a two-component flavin-dependent monooxygenase, the two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome, DNA and amino acid sequence determination and analysis, recombinant expression of His6-tagged PheA2 in Escherichia coli strain M15 | Rhodococcus erythropolis |
| plasmid pQE30A2 expressing His6PheA2 protein transformed into Escherichia coli M15 | Rhodococcus erythropolis |
| plasmid pQE9A1 expressing His6PheA1 protein transformed into Escherichia coli M15 | Rhodococcus erythropolis |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| 4-hydroxymercuribenzoate | complete inhibition at 0.02 mM | Rhodococcus erythropolis |  |
| Ag+ | 0.02 mM completely inhibits; complete inhibition at 0.02 mM | Rhodococcus erythropolis | |
| Co2+ | 1 mM inhibits by 20%; 20% inhibition at 1 mM | Rhodococcus erythropolis | |
| Cu2+ | 0.02 mM completely inhibits; complete inhibition at 0.02 mM | Rhodococcus erythropolis | |
| FAD | with respect to the total phenol hydroxylase activity, concentrations higher than 0.01 mM inhibit the catalyzed reaction; with respect to the total phenol hydroxylase activity, concentrations higher than 0.01 mM inhibit the catalyzed reaction | Rhodococcus erythropolis | |
| Fe2+ | 1 mM inhibits by 49%; 49% inhibition at 1 mM | Rhodococcus erythropolis | |
| Fe3+ | 0.1 mM inhibits by 23%, 1 mM completely inhibits; complete inhibition at 1 mM | Rhodococcus erythropolis | |
| additional information | no effect by 1 mM of iodoacetamide, phenylmethylsulfoxide, or EDTA | Rhodococcus erythropolis | |
| N-ethylmaleimide | 0.1 mM inhibits by 38%, 1 mM completely inhibits; complete inhibition at 1 mM | Rhodococcus erythropolis | |
| Ni2+ | 0.1 mM inhibits by 79%, 1 mM completely inhibits; complete inhibition at 1 mM | Rhodococcus erythropolis | |
| p-hydroxymercuribenzoate | 0.0005 mM inhibits by 53%,0.02 mM completely inhibits the enzymic activity | Rhodococcus erythropolis | |
| Zn2+ | 1 mM inhibits by 77%; 77% inhibition at 1 mM | Rhodococcus erythropolis | |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0134 | - |
FAD | - |
Rhodococcus erythropolis | |
| 0.0533 | - |
NADH | - |
Rhodococcus erythropolis | |
| 0.0677 | - |
riboflavin | - |
Rhodococcus erythropolis | |
| 0.0691 | - |
FMN | - |
Rhodococcus erythropolis | |
| 0.271 | - |
NADPH | with FAD as electron acceptor | Rhodococcus erythropolis | |
| 0.606 | - |
NADPH | with FMN as electron acceptor | Rhodococcus erythropolis | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | 1 mM activates by 27% | Rhodococcus erythropolis | |
| Mg2+ | activates 13% at 1 mM | Rhodococcus erythropolis | |
| Mn2+ | 1 mM activates by 13% | Rhodococcus erythropolis | |
| Mn2+ | activates 27% at 1 mM | Rhodococcus erythropolis | |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 20350 | - |
2 * 20350, sequence analysis, 2 * 22000, SDS-PAGE, 2 * 22550, mass spectrometry | Rhodococcus erythropolis |
| 20350 | - |
2 * 22550, recombinant His6-tagged PheA2, mass spectrometry, 2 * 20350, sequence calculation, 2 * 22000, recombinant His6-tagged PheA2, SDS-PAGE | Rhodococcus erythropolis |
| 22000 | - |
2 * 20350, sequence analysis, 2 * 22000, SDS-PAGE, 2 * 22550, mass spectrometry | Rhodococcus erythropolis |
| 22000 | - |
2 * 22550, recombinant His6-tagged PheA2, mass spectrometry, 2 * 20350, sequence calculation, 2 * 22000, recombinant His6-tagged PheA2, SDS-PAGE | Rhodococcus erythropolis |
| 22550 | - |
2 * 20350, sequence analysis, 2 * 22000, SDS-PAGE, 2 * 22550, mass spectrometry | Rhodococcus erythropolis |
| 22550 | - |
2 * 22550, recombinant His6-tagged PheA2, mass spectrometry, 2 * 20350, sequence calculation, 2 * 22000, recombinant His6-tagged PheA2, SDS-PAGE | Rhodococcus erythropolis |
| 45000 | - |
gel filtration | Rhodococcus erythropolis |
| 45000 | - |
recombinant PheA2, gel filtration | Rhodococcus erythropolis |
| 60720 | - |
4 * 60720, sequence analysis, 4 * 62000, SDS-PAGE, 4 * 62078, mass spectrometry | Rhodococcus erythropolis |
| 60720 | - |
4 * 62078, recombinant His6-tagged PheA1, mass spectrometry, 4 * 60720, sequence calculation, 4 * 62000, recombinant His6-tagged PheA1, SDS-PAGE | Rhodococcus erythropolis |
| 62000 | - |
4 * 60720, sequence analysis, 4 * 62000, SDS-PAGE, 4 * 62078, mass spectrometry | Rhodococcus erythropolis |
| 62000 | - |
4 * 62078, recombinant His6-tagged PheA1, mass spectrometry, 4 * 60720, sequence calculation, 4 * 62000, recombinant His6-tagged PheA1, SDS-PAGE | Rhodococcus erythropolis |
| 62078 | - |
4 * 60720, sequence analysis, 4 * 62000, SDS-PAGE, 4 * 62078, mass spectrometry | Rhodococcus erythropolis |
| 62078 | - |
4 * 62078, recombinant His6-tagged PheA1, mass spectrometry, 4 * 60720, sequence calculation, 4 * 62000, recombinant His6-tagged PheA1, SDS-PAGE | Rhodococcus erythropolis |
| 236000 | - |
gel filtration | Rhodococcus erythropolis |
| 236000 | - |
recombinant PheA1, gel filtration | Rhodococcus erythropolis |
| 238000 | - |
non-denaturing-PAGE followed by staining with Coomassie Brilliant Blue | Rhodococcus erythropolis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| phenol + FADH2 + O2 | Rhodococcus erythropolis | - |
catechol + FAD + H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Rhodococcus erythropolis | A7LCL0 | UPV-1 | - |
| Rhodococcus erythropolis | A7LCL0 | PheA1; gene pheA1 | - |
| Rhodococcus erythropolis | A7LCL1 | UPV-1 | - |
| Rhodococcus erythropolis | A7LCL1 | PheA2; gene pheA2 | - |
| Rhodococcus erythropolis UPV-1 | A7LCL0 | UPV-1 | - |
| Rhodococcus erythropolis UPV-1 | A7LCL0 | PheA1; gene pheA1 | - |
| Rhodococcus erythropolis UPV-1 | A7LCL1 | UPV-1 | - |
| Rhodococcus erythropolis UPV-1 | A7LCL1 | PheA2; gene pheA2 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| on a Ni2+ column, to electrophoretic homogeneity | Rhodococcus erythropolis |
| recombinant His6-tagged PheA1 from Escherichia coli strain M15 | Rhodococcus erythropolis |
| recombinant His6-tagged PheA2 from Escherichia coli strain M15 by nickel affinity chromatography | Rhodococcus erythropolis |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| cell culture | strain UPV-1 is able to grow on phenol as the sole carbon and energy source | Rhodococcus erythropolis | - |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 41.7 | - |
purified recombinant enzyme PheA2, pH 6.8, 30°C | Rhodococcus erythropolis |
| 411.7 | - |
- |
Rhodococcus erythropolis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 3-nitrophenol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis | ? + NAD(P)+ + H2O | - |
? | |
| 3-nitrophenol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis UPV-1 | ? + NAD(P)+ + H2O | - |
? | |
| 4-nitrophenol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis | ? + NAD(P)+ + H2O | - |
? | |
| 4-nitrophenol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis UPV-1 | ? + NAD(P)+ + H2O | - |
? | |
| catechol + FMN + H2O | - |
Rhodococcus erythropolis | phenol + FMNH2 + O2 | - |
r | |
| catechol + riboflavin + H2O | - |
Rhodococcus erythropolis | phenol + reduced riboflavin + O2 | - |
r | |
| additional information | the two-component phenol hydroxylase is completely unable to hydroxylate benzoate, 4-hydroxybenzoate, and orcinol | Rhodococcus erythropolis | ? | - |
? | |
| additional information | the two-component phenol hydroxylase is completely unable to hydroxylate benzoate, 4-hydroxybenzoate, and orcinol | Rhodococcus erythropolis UPV-1 | ? | - |
? | |
| phenol + FADH2 + O2 | - |
Rhodococcus erythropolis | catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | hydroxylation of phenol in vitro requires the presence of both PheA1 and PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction, Km for FAD is 0.0134 mM, Km for NADH is 0.0533 mM. The hydroxylation of phenol in vitro depends on the molar ratio of His6PheA2 and His6PheA1 present in the reaction mixture, an increase of the amount of His6PheA1 in the assay results in a higher phenol hydroxylase activity. In the assay, a reductase/oxygenase molar ratio of 1:10 is used | Rhodococcus erythropolis | catechol + FAD + H2O | - |
? | |
| phenol + FADH2 + O2 | hydroxylation of phenol in vitro requires the presence of both PheA1 and PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction. The hydroxylation of phenol in vitro depends on the molar ratio of His6PheA2 and His6PheA1 present in the reaction mixture, an increase of the amount of His6PheA1 in the assay results in a higher phenol hydroxylase activity. In the assay, a reductase/oxygenase molar ratio of 1:10 is used | Rhodococcus erythropolis | catechol + FAD + H2O | - |
? | |
| phenol + NAD(P)H + H+ + O2 | hydroxylation of phenol in vitro requires the presence of both His6PheA1 and His6PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction | Rhodococcus erythropolis | catechol + NAD(P)+ + H2O | - |
? | |
| phenol + NAD(P)H + H+ + O2 | hydroxylation of phenol in vitro requires the presence of both His6PheA1 and His6PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction | Rhodococcus erythropolis UPV-1 | catechol + NAD(P)+ + H2O | - |
? | |
| phenol + NADH + H+ + O2 | - |
Rhodococcus erythropolis | catechol + NAD+ + H2O | - |
r | |
| phenol + NADH + H+ + O2 | - |
Rhodococcus erythropolis UPV-1 | catechol + NAD+ + H2O | - |
r | |
| resorcinol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis | ? + NAD(P)+ + H2O | - |
? | |
| resorcinol + NAD(P)H + H+ + O2 | - |
Rhodococcus erythropolis UPV-1 | ? + NAD(P)+ + H2O | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | 2 * 20350, sequence analysis, 2 * 22000, SDS-PAGE, 2 * 22550, mass spectrometry | Rhodococcus erythropolis |
| homodimer | 2 * 22550, recombinant His6-tagged PheA2, mass spectrometry, 2 * 20350, sequence calculation, 2 * 22000, recombinant His6-tagged PheA2, SDS-PAGE | Rhodococcus erythropolis |
| homotetramer | 4 * 60720, sequence analysis, 4 * 62000, SDS-PAGE, 4 * 62078, mass spectrometry | Rhodococcus erythropolis |
| homotetramer | 4 * 62078, recombinant His6-tagged PheA1, mass spectrometry, 4 * 60720, sequence calculation, 4 * 62000, recombinant His6-tagged PheA1, SDS-PAGE | Rhodococcus erythropolis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| PheA1 | - |
Rhodococcus erythropolis |
| PheA2 | - |
Rhodococcus erythropolis |
| phenol hydroxylase | - |
Rhodococcus erythropolis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 25 | 40 | assay at | Rhodococcus erythropolis |
| 40 | - |
- |
Rhodococcus erythropolis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 6.8 | - |
- |
Rhodococcus erythropolis |
| 6.8 | - |
assay at | Rhodococcus erythropolis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | - |
Rhodococcus erythropolis | |
| FAD | activity of the oxygenase component His6PheA1 of phenol hydroxylase is strictly dependent on FAD | Rhodococcus erythropolis | |
| FADH2 | - |
Rhodococcus erythropolis | |
| FMN | - |
Rhodococcus erythropolis | |
| additional information | the flavoprotein monooxygenase uses electrons of NAD(P)H to activate and cleave a molecule of oxygen through the formation of an intermediate flavin hydroperoxide and enable the incorporation of an oxygen atom into the substrate | Rhodococcus erythropolis | |
| NADH | - |
Rhodococcus erythropolis | |
| NADH | PheA2 uses NADH in order to reduce FAD, according to a random sequential kinetic mechanism | Rhodococcus erythropolis | |
| NADH | preferred compared to NADPH | Rhodococcus erythropolis | |
| NADPH | - |
Rhodococcus erythropolis | |
| NADPH | can be used instead of NADH as electron donor, using either FAD or FMN as electron acceptor, but with an affinity 5fold or 10fold lower than NADH, respectively | Rhodococcus erythropolis | |
pI Value
| Organism | Comment | pI Value Maximum | pI Value |
|---|---|---|---|
| Rhodococcus erythropolis | - |
- |
5.16 |
| Rhodococcus erythropolis | sequence calculation | - |
5.16 |
| Rhodococcus erythropolis | - |
- |
5.75 |
| Rhodococcus erythropolis | sequence calculation | - |
5.75 |
General Information
| General Information | Comment | Organism |
|---|---|---|
| physiological function | phenol-degrading aerobic bacteria are able to convert phenol into nontoxic intermediates of the tricarboxylic acid cycle via an ortho or meta pathway. The monooxygenation of the aromatic ring constitutes the first step in the biodegradation of many phenolic compounds. The two-component flavin-dependent monooxygenase phenol hydroxylase catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1. Recombinant PheA1 has no phenol hydroxylase activity on its own. Recombinant PheA2 is a flavin reductase that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The hydroxylation of phenol in vitro requires the presence of both PheA1 and PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction. The enzymic activity catalyzed in vitro by His6PheA2 is essential to carry out the hydroxylation of phenol by His6PheA1 | Rhodococcus erythropolis |
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