Cloned (Comment) | Organism |
---|---|
gene pheA2, sequence comparisons, recombinant overexpression of wild-type and selenomethionine-substituted PheA2 in Escherichia coli strain BL21(DE3)pLysS | Parageobacillus thermoglucosidasius |
Crystallization (Comment) | Organism |
---|---|
purified recombinant PheA2 containing bound FAD cofactor and purified reduced holo-PheA2 in complex with oxidized NAD, hanging drop vapor diffusion method, mixing of 0.002 ml of 16 mg/ml protein in 50 mM sodium phosphate buffer, pH 7.0, with 0.002 ml of reservoir solution containing 20-26% PEG 3350 and 0.4 M magnesium nitrate, and equilibration against 1 ml of reservoir solution, 20°C, several days, crystal soakig in NADH or FAD solution, X-ray diffraction structure determination and analysis at 2.1-2.2 A resolution, modeling | Parageobacillus thermoglucosidasius |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
phenol + FADH2 + O2 | Parageobacillus thermoglucosidasius | a two-component enzyme system: the smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols | catechol + FAD + H2O | - |
? | |
phenol + FADH2 + O2 | Parageobacillus thermoglucosidasius A7 | a two-component enzyme system: the smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols | catechol + FAD + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Parageobacillus thermoglucosidasius | - |
or Bacillus thermoglucosidasius, gene pheA2 | - |
Parageobacillus thermoglucosidasius A7 | - |
or Bacillus thermoglucosidasius, gene pheA2 | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and selenomethionine-substituted PheA2 from Escherichia coli strain BL21(DE3)pLysS by protamine sulfate fractionation, hydrophobic interaction chromatography, dialysis, anion exchange chromatography, and preparative gel filtration | Parageobacillus thermoglucosidasius |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
phenol + FADH2 + O2 = catechol + FAD + H2O | smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping pong bisubstrate-biproduct mechanism | Parageobacillus thermoglucosidasius |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
phenol + FADH2 + O2 | a two-component enzyme system: the smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols | Parageobacillus thermoglucosidasius | catechol + FAD + H2O | - |
? | |
phenol + FADH2 + O2 | reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. PheA2 is able to bind one FAD cofactor and one FAD substrate | Parageobacillus thermoglucosidasius | catechol + FAD + H2O | - |
? | |
phenol + FADH2 + O2 | a two-component enzyme system: the smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols | Parageobacillus thermoglucosidasius A7 | catechol + FAD + H2O | - |
? | |
phenol + FADH2 + O2 | reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. PheA2 is able to bind one FAD cofactor and one FAD substrate | Parageobacillus thermoglucosidasius A7 | catechol + FAD + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis | Parageobacillus thermoglucosidasius |
Synonyms | Comment | Organism |
---|---|---|
flavin reductase PheA2 | - |
Parageobacillus thermoglucosidasius |
PheA2 | - |
Parageobacillus thermoglucosidasius |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Parageobacillus thermoglucosidasius |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Parageobacillus thermoglucosidasius |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FADH2 | PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent, binding structure, overview | Parageobacillus thermoglucosidasius | |
additional information | reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. PheA2 is able to bind one FAD cofactor and one FAD substrate. PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis. No activity with FMN, riboflavin, and NADPH | Parageobacillus thermoglucosidasius | |
NADH | addition of NADH to crystalline PheA2 reduces the flavin cofactor, the NAD product is bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking, binding structure, overview | Parageobacillus thermoglucosidasius |
General Information | Comment | Organism |
---|---|---|
additional information | structure analysis and modeling | Parageobacillus thermoglucosidasius |
physiological function | the catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a pingpong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols | Parageobacillus thermoglucosidasius |