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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

Summary:
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 1.13.11.4) and 1-hydroxy-2-naphthoate dioxygenase (EC 1.13.11.38). 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: ?

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


Enzymatic reaction of: salicylate 1,2-dioxygenase

EC Number: 1.13.11.-

salicylate + oxygen <=> 2-oxohepta--3,5-dienedioate + H+

The reaction direction shown, that is, A + B ↔ C + D versus C + D ↔ A + B, 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 ]

Summary:
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]


References

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


Report Errors or Provide Feedback
Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
Page generated by SRI International Pathway Tools version 18.5 on Tue Nov 25, 2014, biocyc13.