Literature summary for 1.14.14.20 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.

Search for more literature for 1.14.14.20

Cloned(Commentary)

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 (Commentary)

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

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/ Products (Substrates)

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

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

Purification (Commentary)

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

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	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
flavin reductase PheA2	-	Parageobacillus thermoglucosidasius
PheA2	-	Parageobacillus thermoglucosidasius

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
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

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