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Literature summary extracted from

  • Nakano, H.; Wieser, M.; Hurh, B.; Kawai, T.; Yoshida, T.; Yamane, T.; Nagasawa, T.
    Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5 (1999), Eur. J. Biochem., 260, 120-126.
    View publication on PubMed

Application

EC Number Application Comment Organism
1.14.13.114 synthesis the isolated enzyme is used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy Pseudomonas fluorescens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.13.114 expression in Escherichia coli Pseudomonas fluorescens

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.14.13.114 5,5'-dithiobis(2-nitrobenzoate) 1 mM, complete inhibition Pseudomonas fluorescens
1.14.13.114 Ag2SO4 1 mM, complete inhibition Pseudomonas fluorescens
1.14.13.114 CuCl2 1 mM, complete inhibition Pseudomonas fluorescens
1.14.13.114 HgCl2 1 mM, complete inhibition Pseudomonas fluorescens
1.14.13.114 additional information no significant effect on enzyme activity is found with metal-chelating agents such o-phenanthroline, 8-hydroxyquinoline, EDTA, disodium 4,5-dihydroxy-m-benzenedisulfonate, fluoride and azide, and other compounds such as KCl, LiCl, NaCl, BaCl2, CaCl2, MnCl2, MgCl2, PbCl2, ZnCl2, CoCl2, SnCl2, FeSO4, FeCl3, NiCl2, CdCl2, AlCl3, iodoacetic acid, hydroxylamine, phenylhydrazine, semicarbazide, cysteamine, alpha,alpha'-dipyridyl and urea Pseudomonas fluorescens
1.14.13.114 N-ethylmaleimide 1 mM, 69% inhibition Pseudomonas fluorescens
1.14.13.114 nicotinate potent competitive inhibitor Pseudomonas fluorescens
1.14.13.114 p-chloromercuribenzoate 1 mM, complete inhibition Pseudomonas fluorescens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.14.13.114 0.098
-
 6-Hydroxynicotinate pH 7.0, 30°C Pseudomonas fluorescens
1.14.13.114 0.15
-
 4-hydroxybenzoate pH 7.0, 30°C Pseudomonas fluorescens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
1.14.13.114 membrane bound to Pseudomonas fluorescens 16020
-

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.14.13.114 42886
-
 1 * 42886, including the starting methionine, calculated from sequence Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 Pseudomonas fluorescens aerobic catabolism of nicotinic acid. NADH is 5times more effective than NADPH 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 Pseudomonas fluorescens TN5 aerobic catabolism of nicotinic acid. NADH is 5times more effective than NADPH 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.13.114 Pseudomonas fluorescens
-
-
-
1.14.13.114 Pseudomonas fluorescens TN5
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.14.13.114
-
 Pseudomonas fluorescens

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.14.13.114 6.67
-
-
 Pseudomonas fluorescens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.13.114 4-hydroxybenzoate + NADH + H+ + O2 6.0% relative activity compared with 6-hydroxynicotinate Pseudomonas fluorescens hydroquinone + NAD+ + H2O + CO2
-
 ?
1.14.13.114 4-hydroxybenzoate + NADH + H+ + O2 6.0% relative activity compared with 6-hydroxynicotinate Pseudomonas fluorescens TN5 hydroquinone + NAD+ + H2O + CO2
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 aerobic catabolism of nicotinic acid. NADH is 5times more effective than NADPH Pseudomonas fluorescens 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 NADH is 5times more effective than NADPH Pseudomonas fluorescens 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 aerobic catabolism of nicotinic acid. NADH is 5times more effective than NADPH Pseudomonas fluorescens TN5 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 NADH is 5times more effective than NADPH Pseudomonas fluorescens TN5 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.14.13.114 additional information 3-hydroxybenzoate (0.49% relative activity compared with 6-hydroxynicotinate), 2-hydroxybenzoate (0.18% compared with 6-hydroxynicotinate), 2-hydroxynicotinate (0.31% relative activity compared with 6-hydroxynicotinate) and 6-hydroxypyrazine carboxylate (0.19% relative activity compared with 6-hydroxynicotinate) are less effecive substrates or, in the case of nicotinate, 6-methylnicotinate and benzoate, not substrates at all Pseudomonas fluorescens ?
-
 ?
1.14.13.114 additional information 3-hydroxybenzoate (0.49% relative activity compared with 6-hydroxynicotinate), 2-hydroxybenzoate (0.18% compared with 6-hydroxynicotinate), 2-hydroxynicotinate (0.31% relative activity compared with 6-hydroxynicotinate) and 6-hydroxypyrazine carboxylate (0.19% relative activity compared with 6-hydroxynicotinate) are less effecive substrates or, in the case of nicotinate, 6-methylnicotinate and benzoate, not substrates at all Pseudomonas fluorescens TN5 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
1.14.13.114 monomer 1 * 40000-42000, SDS-PAGE Pseudomonas fluorescens
1.14.13.114 monomer 1 * 42886, including the starting methionine, calculated from sequence Pseudomonas fluorescens

Synonyms

EC Number Synonyms Comment Organism
1.14.13.114 6-hydroxynicotinic acid 3-monooxygenase
-
 Pseudomonas fluorescens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.14.13.114 35
-
-
 Pseudomonas fluorescens

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.14.13.114 35
-
 pH 7.0, 10 min, in the absence of FAD, apoenzyme is stable below Pseudomonas fluorescens
1.14.13.114 40
-
 pH 7.0, 10 min, in the presence of 0.5 mM FAD, holoenzyme is stable below Pseudomonas fluorescens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.14.13.114 7.6
-
-
 Pseudomonas fluorescens

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.14.13.114 6 9 35°C, 10 min, stable between pH 6.0 and pH 9.0 Pseudomonas fluorescens

Cofactor

EC Number Cofactor Comment Organism Structure
1.14.13.114 FAD the enzyme activity is NADH-dependent and FAD-dependent. The holoenzyme contains 1 M of FAD per 1 M of enzyme. FAD gradually dissociates from the enzyme during purification. Without FAD, no pure enzyme activity is observed, but after the addition of FAD, the apoenzyme is activated immediately Pseudomonas fluorescens
1.14.13.114 additional information riboflavin or FMN do not serve as enzyme cofactors Pseudomonas fluorescens
1.14.13.114 NADH the enzyme activity is NADH-dependent and FAD-dependent. NADH is 5times more effective than NADPH Pseudomonas fluorescens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.14.13.114 0.49
-
 nicotinate
-
 Pseudomonas fluorescens