BRENDA - Enzyme Database

Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Fang, T.; Zhou, N.Y.; Appl. Microbiol. Biotechnol. 98, 671-679 (2014)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.14.13.172
expressed in Escherichia coli BL21(DE3) cells
Ralstonia sp.
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.13.172
0.05641
-
NADPH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.05976
-
NADH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.1071
-
salicylate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.3284
-
2-Nitrophenol
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.9398
-
2,6-dihydroxybenzoate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.13.172
Iron
the enzyme contains one Rieske-type [2Fe-2S] cluster and 7.5 g iron per mol enzyme
Ralstonia sp.
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.14.13.172
18800
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
1.14.13.172
20000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
1.14.13.172
51000
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
1.14.13.172
52000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
1.14.13.172
195200
-
gel filtration
Ralstonia sp.
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.13.172
salicylate + NADH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.14.13.172
Ralstonia sp.
O52379 and O52380
large oxygenase component and small oxagenase component
-
1.14.13.172
Ralstonia sp. U2
O52379 and O52380
large oxygenase component and small oxagenase component
-
Purification (Commentary)
EC Number
Commentary
Organism
1.14.13.172
the three-component Fe-S protein complex (NagAaGHAb) of the enzyme is purified by HisTrap column chromatography and Sephadex G-25 gel filtration
Ralstonia sp.
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.14.13.172
107.1
-
after 124fold purification, in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.13.172
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.14.13.172
heterohexamer
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence; 3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.14.13.172
NADH
-
Ralstonia sp.
1.14.13.172
NADPH
-
Ralstonia sp.
1.14.13.172
[2Fe-2S]-center
-
Ralstonia sp.
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.13.172
expressed in Escherichia coli BL21(DE3) cells
Ralstonia sp.
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.14.13.172
NADH
-
Ralstonia sp.
1.14.13.172
NADPH
-
Ralstonia sp.
1.14.13.172
[2Fe-2S]-center
-
Ralstonia sp.
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.14.13.172
0.05641
-
NADPH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.05976
-
NADH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.1071
-
salicylate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.3284
-
2-Nitrophenol
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
1.14.13.172
0.9398
-
2,6-dihydroxybenzoate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
1.14.13.172
Iron
the enzyme contains one Rieske-type [2Fe-2S] cluster and 7.5 g iron per mol enzyme
Ralstonia sp.
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.14.13.172
18800
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
1.14.13.172
20000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
1.14.13.172
51000
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
1.14.13.172
52000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
1.14.13.172
195200
-
gel filtration
Ralstonia sp.
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.14.13.172
salicylate + NADH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.14.13.172
the three-component Fe-S protein complex (NagAaGHAb) of the enzyme is purified by HisTrap column chromatography and Sephadex G-25 gel filtration
Ralstonia sp.
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
1.14.13.172
107.1
-
after 124fold purification, in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.14.13.172
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
1.14.13.172
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
1.14.13.172
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.14.13.172
heterohexamer
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence; 3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.