BRENDA - Enzyme Database
show all sequences of 1.14.13.172

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)
Commentary
Organism
expressed in Escherichia coli BL21(DE3) cells
Ralstonia sp.
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.05641
-
NADPH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.05976
-
NADH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.1071
-
salicylate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.3284
-
2-Nitrophenol
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.9398
-
2,6-dihydroxybenzoate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Iron
the enzyme contains one Rieske-type [2Fe-2S] cluster and 7.5 g iron per mol enzyme
Ralstonia sp.
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
18800
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
20000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
51000
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
52000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
195200
-
gel filtration
Ralstonia sp.
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
salicylate + NADH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
salicylate + NADH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
salicylate + NADPH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
salicylate + NADPH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Ralstonia sp.
O52379 and O52380
large oxygenase component and small oxagenase component
-
Ralstonia sp. U2
O52379 and O52380
large oxygenase component and small oxagenase component
-
Purification (Commentary)
Commentary
Organism
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]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
107.1
-
after 124fold purification, in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
Subunits
Subunits
Commentary
Organism
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
Cofactor
Commentary
Organism
Structure
NADH
-
Ralstonia sp.
NADPH
-
Ralstonia sp.
[2Fe-2S]-center
-
Ralstonia sp.
Cloned(Commentary) (protein specific)
Commentary
Organism
expressed in Escherichia coli BL21(DE3) cells
Ralstonia sp.
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADH
-
Ralstonia sp.
NADPH
-
Ralstonia sp.
[2Fe-2S]-center
-
Ralstonia sp.
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.05641
-
NADPH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.05976
-
NADH
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.1071
-
salicylate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.3284
-
2-Nitrophenol
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
0.9398
-
2,6-dihydroxybenzoate
in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
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)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
18800
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
20000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
51000
-
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence
Ralstonia sp.
52000
-
3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
195200
-
gel filtration
Ralstonia sp.
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
salicylate + NADH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
salicylate + NADH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
?
salicylate + NADPH + H+ + O2
Ralstonia sp.
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
salicylate + NADPH + H+ + O2
Ralstonia sp. U2
-
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
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)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
107.1
-
after 124fold purification, in 50 mM MES buffer (pH 6.4), at 22°C
Ralstonia sp.
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2,4-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2,6-dihydroxybenzoate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp.
?
-
-
-
?
2-nitrophenol + NADH + H+ + O2
-
721398
Ralstonia sp. U2
?
-
-
-
?
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
salicylate + NADH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NAD+ + H2O
-
-
-
?
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp.
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
salicylate + NADPH + H+ + O2
-
721398
Ralstonia sp. U2
2,5-dihydroxybenzoate + NADP+ + H2O
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
heterohexamer
3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence; 3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE
Ralstonia sp.
Other publictions for EC 1.14.13.172
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
743932
Sun
Precursor-directed biosynthes ...
Cupriavidus necator
ACS Synth. Biol.
4
554-558
2015
-
-
1
-
1
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
721398
Fang
Purification and characterizat ...
Ralstonia sp., Ralstonia sp. U2
Appl. Microbiol. Biotechnol.
98
671-679
2014
-
-
1
-
-
-
-
5
-
1
5
4
-
6
-
-
1
-
-
-
1
-
10
1
-
-
-
-
-
-
-
3
-
-
-
-
-
1
3
-
-
-
-
-
-
5
-
1
5
4
-
-
-
1
-
-
1
-
10
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
722507
Zhou
Salicylate 5-hydroxylase from ...
Ralstonia sp., Ralstonia sp. U2
J. Bacteriol.
184
1547-1555
2002
-
-
1
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
722496
Fuenmayor
A gene cluster encoding steps ...
Pseudomonas sp., Pseudomonas sp. U2
J. Bacteriol.
180
2522-2530
1998
-
-
1
-
-
-
-
-
-
-
2
4
-
6
-
-
-
-
-
-
-
-
4
1
-
-
-
-
-
-
-
3
-
-
-
-
-
1
3
-
-
-
-
-
-
-
-
-
2
4
-
-
-
-
-
-
-
-
4
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
438959
Suemori
-
Physicochemical and immunochem ...
Rhodococcus erythropolis, Rhodococcus erythropolis S1
Biotechnol. Lett.
17
1063-1068
1995
-
-
-
-
-
-
-
-
-
-
2
-
-
5
-
-
1
-
-
-
-
-
4
1
-
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
2
-
-
-
-
1
-
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-
-
4
1
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-
-
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-
-
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