MetaCyc Enzyme: salicylate 1,2-dioxygenase

Gene: AY323951 Accession Number: G-12243 (MetaCyc)

Species: Pseudaminobacter salicylatoxidans BN12

Subunit composition of salicylate 1,2-dioxygenase = [AY323951]4
         salicylate 1,2-dioxygenase subunit = AY323951

The native apparent molecular mass was determined by gel filtration chromatography [Hintner01].

This enzyme from the naphthalenesulfonate-degrading bacterium Pseudaminobacter salicylatoxidans BN12 catalyzes a direct salicylate ring fission to produce 2-oxohepta--3,5-dienedioate. The native enzyme was purified and characterized as a homotetramer [Hintner01]. Recombinant, His-tagged enzyme was expressed in Escherichia coli, purified and characterized [Hintner04].

This enzyme was unique in its ability to efficiently cleave a broad range of salicylates, gentisates and 1-hydroxy-2-naphthoate in comparison with the more substrate-specific gentisate 1,2-dioxygenases (EC and 1-hydroxy-2-naphthoate dioxygenase (EC With the exception of 1-hydroxy-2-naphthoate dioxygenase, this enzyme differed from other ring fission dioxygenases in its ability to cleave substituted salicylates carrying a single hydroxy group. Such substrates are not activated for ring fission by other electron-donating substituents. It was also different from known gentisate 1,2-dioxygenases, or 1-hydroxy-2-naphthoate dioxygenase, in its ability to cleave salicylate, as well as its ability to efficiently cleave gentisate and 1-hydroxy-2-naphthoate. Gentisate 1,2-dioxygenases do not oxidize salicylate and 1-hydroxy-2-naphthoate dioxygenase does not oxidize salicylate or gentisate (in [Hintner04] and in [Matera08]).

Amino acid sequence comparisons showed 31% sequence identity between this enzyme and a gentisate 1,2-dioxygenase and 28% identity for a 1-hydroxy-2-naphthoate dioxygenase, suggesting that they are only distantly related to this enzyme [Hintner04].

The crystal structure of this homotetrameric enzyme has been determined at 2.9 Å resolution. It is a member of the type III extradiol-type dioxygenases. The catalytic center contained a mononuclear iron(II) coordinated to three histidine residues within the N-terminal domain. Active site residues responsible for the broad substrate specificity were identified [Matera08].
The subunit apparent molecular mass was determined by SDS-PAGE [Hintner01].
The gene has not been named and is referred to here by accession number.

Molecular Weight of Polypeptide: 41.106 kD (from nucleotide sequence), 45.0 kD (experimental) [Hintner01]

Molecular Weight of Multimer: 185.0 kD (experimental) [Hintner01]

Unification Links: Entrez-Nucleotide:AY323951, Protein Model Portal:Q67FT0, SMR:Q67FT0, UniProt:Q67FT0

Relationship Links: InterPro:IN-FAMILY:IPR011051, InterPro:IN-FAMILY:IPR013096, InterPro:IN-FAMILY:IPR014710, PDB:Structure:2PHD, PDB:Structure:3NJZ, PDB:Structure:3NKT, PDB:Structure:3NL1, PDB:Structure:3NST, PDB:Structure:3NVC, PDB:Structure:3NW4, PDB:Structure:4FAG, PDB:Structure:4FAH, PDB:Structure:4FBF, Pfam:IN-FAMILY:PF07883

Gene-Reaction Schematic

Gene-Reaction Schematic

Created 06-Oct-2010 by Fulcher CA, SRI International

Enzymatic reaction of: salicylate 1,2-dioxygenase

Inferred from experiment

EC Number: 1.13.11.-

salicylate + oxygen → 2-oxohepta--3,5-dienedioate + H+

The direction shown, i.e. which substrates are on the left and right sides, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

Alternative Substrates for salicylate: 5-aminosalicylate [Hintner01], gentisate [Hintner01], 1-hydroxy-2-naphthoate [Hintner01]

This 1,2-dioxygenase catalyzes a direct fission of the salicylate ring in a reaction that is oxygen-dependent, but independent of NAD(P)H. The product of the reaction was identified as 2-oxohepta--3,5-dienedioate by mass spectrometry and 1H and 13C NMR spectroscopy [Hintner01].

In assays of cell extracts the enzyme was inhibited by Fe2+ chelators, suggesting Fe2+ as a cofactor [Hintner01].

For the purified enzyme, the higher Kcat/Km values for gentisate and 1-hydroxy-2-naphthoate suggested that these substrates were preferred over salicylate and the enzyme was concluded to be a gentisate 1,2-dioxygenase with a very broad substrate range [Hintner01].

The alternative substrates 5-aminosalicylate, gentisate and 1-hydroxy-2-naphthoate were determined for the purified native enzyme [Hintner01] and the purified recombinant enzyme [Hintner04]. The purified recombinant enzyme also utilized 3-, 4-, and 5-substituted methyl-, hydroxy-, amino-, fluoro-, chloro- and bromo-substituted salicylates and 3,5-dichloro and 3.5-dibromo-substituted salicylates. The corresponding substituted 2-oxohepta-3,5-dienoate products were identified [Hintner04].

Cofactors or Prosthetic Groups: Fe2+ [Hintner01]

Inhibitors (Unknown Mechanism): 2,2'-dipyridyl [Hintner01], 8-hydroxyquinoline [Hintner01], o-phenanthroline [Hintner01]Kinetic Parameters:
Substrate Km (μM) Citations
salicylate 12.0 [Hintner01]

T(opt): 40 °C [Hintner01]


Hintner01: Hintner JP, Lechner C, Riegert U, Kuhm AE, Storm T, Reemtsma T, Stolz A (2001). "Direct ring fission of salicylate by a salicylate 1,2-dioxygenase activity from Pseudaminobacter salicylatoxidans." J Bacteriol 183(23);6936-42. PMID: 11698383

Hintner04: Hintner JP, Reemtsma T, Stolz A (2004). "Biochemical and molecular characterization of a ring fission dioxygenase with the ability to oxidize (substituted) salicylate(s) from Pseudaminobacter salicylatoxidans." J Biol Chem 279(36);37250-60. PMID: 15220336

Matera08: Matera I, Ferraroni M, Burger S, Scozzafava A, Stolz A, Briganti F (2008). "Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase." J Mol Biol 380(5);856-68. PMID: 18572191

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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