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Literature summary extracted from

  • van den Heuvel, R.H.; Westphal, A.H.; Heck, A.J.; Walsh, M.A.; Rovida, S.; van Berkel, W.J.; Mattevi, A.
    Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action (2004), J. Biol. Chem., 279, 12860-12867.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.14.20 gene pheA2, sequence comparisons, recombinant overexpression of wild-type and selenomethionine-substituted PheA2 in Escherichia coli strain BL21(DE3)pLysS Parageobacillus thermoglucosidasius

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.14.20 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/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.14.20 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
-
?
1.14.14.20 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

EC Number Organism UniProt Comment Textmining
1.14.14.20 Parageobacillus thermoglucosidasius
-
or Bacillus thermoglucosidasius, gene pheA2
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1.14.14.20 Parageobacillus thermoglucosidasius A7
-
or Bacillus thermoglucosidasius, gene pheA2
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.14.20 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

EC Number Reaction Comment Organism Reaction ID
1.14.14.20 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 and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.14.20 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
-
?
1.14.14.20 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
-
?
1.14.14.20 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
-
?
1.14.14.20 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

EC Number Subunits Comment Organism
1.14.14.20 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

EC Number Synonyms Comment Organism
1.14.14.20 flavin reductase PheA2
-
Parageobacillus thermoglucosidasius
1.14.14.20 PheA2
-
Parageobacillus thermoglucosidasius

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.14.20 25
-
assay at Parageobacillus thermoglucosidasius

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.14.20 7
-
assay at Parageobacillus thermoglucosidasius

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.14.20 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
1.14.14.20 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
1.14.14.20 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

EC Number General Information Comment Organism
1.14.14.20 additional information structure analysis and modeling Parageobacillus thermoglucosidasius
1.14.14.20 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