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Literature summary extracted from

  • Costa, D.M.A.; Gomez, S.V.; de Araujo, S.S.; Pereira, M.S.; Alves, R.B.; Favaro, D.C.; Hengge, A.C.; Nagem, R.A.P.; Brandao, T.A.S.
    Catalytic mechanism for the conversion of salicylate into catechol by the flavin-dependent monooxygenase salicylate hydroxylase (2019), Int. J. Biol. Macromol., 129, 588-600 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.1 gene nahG, recombinant expression of His6-tagged enzyme in Escherichia coli strain Rosetta (DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.14.13.1 purified recombinant detagged apoform of NahG, hanging drop vapor diffusion method, crystallization from reservoir solution containing 1.5 M ammonium sulfate, 0.1 M Tris, pH 8.5, and 12% v/v glycerol, method optimization, 18°C, cryoprotectant solution is formed by the reservoir solution supplemented with 10% v/v ethylene glycol, and 0.06 M sodium iodide, X-ray diffraction structure determination and analysis at 2.0 A resolution, structure modeling using the structure with PDB ID 6BZ5 as a template Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.1 additional information
-
 additional information kinetic analysis, rate-limiting steps, detailed overview Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.13.1 NaCl activates at an ionic strength of 0.222 Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.1 salicylate + NADH + 2 H+ + O2 Pseudomonas putida
-
 catechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 salicylate + NADH + 2 H+ + O2 Pseudomonas putida G7
-
 catechol + NAD+ + H2O + CO2
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.1 Pseudomonas putida Q8RMN4
-
-
1.14.13.1 Pseudomonas putida G7 Q8RMN4
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.1 recombinant expression of His6-tagged enzyme from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography, tag cleavage and a second step of nickel affinity chromatography Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.14.13.1 salicylate + NADH + H+ + O2 = catechol + NAD+ + H2O + CO2 oxidative decarboxylation of salicylate catalyzed by NahG, catalytic mechanism, detailed overview. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis Pseudomonas putida
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.1 3-methylsalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida 3-methylcatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 3-methylsalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida G7 3-methylcatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 5-aminosalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida 5-aminocatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 5-aminosalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida G7 5-aminocatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 5-methylsalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida 5-methylcatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 5-methylsalicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida G7 5-methylcatechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 additional information product identification by NMR Pseudomonas putida ?
-
-
1.14.13.1 additional information product identification by NMR Pseudomonas putida G7 ?
-
-
1.14.13.1 salicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida catechol + NAD+ + H2O + CO2
-
 r
1.14.13.1 salicylate + NADH + 2 H+ + O2
-
 Pseudomonas putida G7 catechol + NAD+ + H2O + CO2
-
 r

Subunits

EC Number Subunits Comment Organism
1.14.13.1 monomer
-
 Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.14.13.1 flavin-dependent monooxygenase salicylate hydroxylase
-
 Pseudomonas putida
1.14.13.1 NahG
-
 Pseudomonas putida
1.14.13.1 salicylate hydroxylase
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.1 25
-
 assay at Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.1 8.5
-
 assay at Pseudomonas putida

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.14.13.1 3.5 11 activity range, profile overview Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.1 FAD required, binding structure analysis. The rearrangement of FAD between the out and in positions is required in the catalytic mechanism comprised of reductive and oxidative reactions, which are shared among NahG and other flavin-dependent monooxygenases Pseudomonas putida
1.14.13.1 NAD+
-
 Pseudomonas putida
1.14.13.1 NADH
-
 Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.14.13.1 evolution the enzyme reaction links the upper and lower pathways of naphthalene degradation by soil pseudomonads, a bacterial genus encompassing many species that can use naphthalene or salicylate as sole carbon sources. NahG hydroxylates and decarboxylates the substrate at the same aromatic carbon atom (ipso substitution), an exquisite feature that is remarkable from a synthetic perspective. NahG is one of the few examples of a flavin enzyme that catalyzes an ortho hydroxylation relative to the substrate's phenol group, which is usually required for efficient catalysis. Other monooxygenases exhibit different regioselectivities. Enzyme structure comparisons, overview. The aromatic residues F85,W87, F230, F243, and W293 are positioned on the antiparallel beta-sheet opposite from the FAD isoalloxazine ring and likely form a hydrophobic environment facing the salicylate. In addition, a number of nonpolar amino acid residues, including A190, M194, M219, L221, L228, and V241, also compose the substrate-binding pocket, whereas the charged residues D224, H226, R247, H322, E381, and R383 are distinctly grouped near the active site, laterally positioned on the re-side of the isoalloxazine and leading to the solvent-accessible protein surface Pseudomonas putida
1.14.13.1 metabolism salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida strain G7 with stoichiometric consumption of NADH and O2. This reaction links the upper and lower pathways of naphthalene degradation by soil pseudomonads, a bacterial genus encompassing many species that can use naphthalene or salicylate as sole carbon sources Pseudomonas putida
1.14.13.1 additional information struccture-function relationship and reaction mechansim analysis, overview. Hammett plots for Km and kcat using substituted salicylates indicate changes in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups Pseudomonas putida
1.14.13.1 physiological function salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida strain G7 Pseudomonas putida

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.14.13.1 100000
-
 salicylate above, recombinant holoenzyme, pH 8.5, 25°C Pseudomonas putida