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Literature summary extracted from

  • Fang, T.; Zhou, N.Y.
    Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2 (2014), Appl. Microbiol. Biotechnol., 98, 671-679.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) 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 Comment 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 Comment 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] Comment 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 Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
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 UniProt Comment 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 Purification (Comment) 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] Comment 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 Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.172 2,4-dihydroxybenzoate + NADH + H+ + O2
-
 Ralstonia sp. ?
-
 ?
1.14.13.172 2,4-dihydroxybenzoate + NADH + H+ + O2
-
 Ralstonia sp. U2 ?
-
 ?
1.14.13.172 2,6-dihydroxybenzoate + NADH + H+ + O2
-
 Ralstonia sp. ?
-
 ?
1.14.13.172 2,6-dihydroxybenzoate + NADH + H+ + O2
-
 Ralstonia sp. U2 ?
-
 ?
1.14.13.172 2-nitrophenol + NADH + H+ + O2
-
 Ralstonia sp. ?
-
 ?
1.14.13.172 2-nitrophenol + NADH + H+ + O2
-
 Ralstonia sp. U2 ?
-
 ?
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
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.172 heterohexamer 3 * 51000 + 3 * 18800, His6-tagged enzyme, calculated from amino acid sequence Ralstonia sp.
1.14.13.172 heterohexamer 3 * 52000 + 3 * 20000, His6-tagged enzyme, SDS-PAGE Ralstonia sp.

Synonyms

EC Number Synonyms Comment Organism
1.14.13.172 NagGH
-
 Ralstonia sp.
1.14.13.172 S5H
-
 Ralstonia sp.

Cofactor

EC Number Cofactor Comment 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.