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Literature summary extracted from

  • Diaz-Saez, L.; Srikannathasan, V.; Zoltner, M.; Hunter, W.N.
    Structures of bacterial kynurenine formamidase reveal a crowded binuclear zinc catalytic site primed to generate a potent nucleophile (2014), Biochem. J., 462, 581-589.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.9 gene kynB, recombinant expression of the His6-tagged enzyme in Escherichia coli K12 strain BL21(DE3)pLysS, codon optimization Pseudomonas aeruginosa
3.5.1.9 gene kynB, recombinant expression of the His6-tagged enzyme in Escherichia coli K12 strain BL21(DE3)pLysS, codon optimization Burkholderia cenocepacia
3.5.1.9 gene kynB, sequence comparison, recombinant expression of the His6-tagged enzyme in Escherichia coli K12 strain BL21(DE3)pLysS, codon optimization Bacillus anthracis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.9 purified detagged recombinant enzyme, hanging drop vapour diffusion method, mixing of 4.0 mg/ml protein in 20 mM Tris-HCl, pH 7.4, and 200 mM NaCl, and 8 mM kynurenine, in a 1.1 ratio with reservoir solution containing 100 mM Tris-HCl, pH 8.5, 150 mM MgCl2, 30% w/v PEG 4000 and 1.5% v/v dioxane, final volume 0.002-0.004 ml, 20°C, 5 days, X-ray diffraction structure determination and analysis at 1.95 A resolution Bacillus anthracis
3.5.1.9 purified detagged recombinant enzyme, sitting drop vapour diffusion method, mixing of 7.5 mg/ml protein in 25 mM Tris-HCl, pH 7.5, and 100 mM NaCl, and kynurenine, in a 1.1 ratio with reservoir solution containing 0.1M HEPES, pH 7.5, 20% w/v PEG 4000 and 10% v/v propan-2-ol, final volume 0.002-0.004 ml, 5 days at room temperature, X-ray diffraction structure determination and analysis at 2.37 A resolution, molecular replacement Pseudomonas aeruginosa
3.5.1.9 purified detagged recombinant enzyme, sitting drop vapour diffusion method, mixing of 7.5 mg/ml protein in 25 mM Tris-HCl, pH 7.5, and 100 mM NaCl, and kynurenine, in a 1.1 ratio with reservoir solution containing 10-16% w/v PEG 3350, 5 mM CoCl2, 5 mM CdCl2, 5 mM MgCl2 and 5 mM NiCl2, final volume 0.002-0.004 ml, 5 days at room temperature, X-ray diffraction structure determination and analysis at 1.60 A resolution Burkholderia cenocepacia

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.9 2-aminoacetophenone active site of the BaKynB-2-aminoacetophenone complex, structure, overview Bacillus anthracis
3.5.1.9 2-aminoacetophenone binding structure, overview Burkholderia cenocepacia
3.5.1.9 2-aminoacetophenone binding structure, overview Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.9 additional information
-
 additional information Michaelis-Menten kinetics Pseudomonas aeruginosa
3.5.1.9 additional information
-
 additional information Michaelis-Menten kinetics Burkholderia cenocepacia
3.5.1.9 additional information
-
 additional information Michaelis-Menten kinetics Bacillus anthracis
3.5.1.9 0.4
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Bacillus anthracis
3.5.1.9 0.57
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Burkholderia cenocepacia
3.5.1.9 0.98
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Pseudomonas aeruginosa

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.1.9 Cd2+ active site-bound Burkholderia cenocepacia
3.5.1.9 additional information cations occupy a crowded environment, and, unlike most Zn2+ -dependent enzymes, there is little scope to increase co-ordination number during catalysis Pseudomonas aeruginosa
3.5.1.9 additional information cations occupy a crowded environment, and, unlike most Zn2+ -dependent enzymes, there is little scope to increase co-ordination number during catalysis Bacillus anthracis
3.5.1.9 additional information presence of Cd2+ and Zn2+ in the active site of, cations occupy a crowded environment, and, unlike most Zn2+ -dependent enzymes, there is little scope to increase co-ordination number during catalysis Burkholderia cenocepacia
3.5.1.9 Zn2+ required, the enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site Pseudomonas aeruginosa
3.5.1.9 Zn2+ required, the enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site Burkholderia cenocepacia
3.5.1.9 Zn2+ required, the enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site Bacillus anthracis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.1.9 40000
-
 2 * 40000, about, recombinant enzyme, SDS-PAGE Pseudomonas aeruginosa
3.5.1.9 40000
-
 2 * 40000, about, recombinant enzyme, SDS-PAGE Burkholderia cenocepacia
3.5.1.9 40000
-
 2 * 40000, about, recombinant enzyme, SDS-PAGE Bacillus anthracis
3.5.1.9 80000
-
 about, recombinant enzyme, native PAGE Pseudomonas aeruginosa
3.5.1.9 80000
-
 about, recombinant enzyme, native PAGE Burkholderia cenocepacia
3.5.1.9 80000
-
 about, recombinant enzyme, native PAGE Bacillus anthracis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.9 N-formyl-L-kynurenine + H2O Pseudomonas aeruginosa
-
 formate + L-kynurenine
-
 ?
3.5.1.9 N-formyl-L-kynurenine + H2O Burkholderia cenocepacia
-
 formate + L-kynurenine
-
 ?
3.5.1.9 N-formyl-L-kynurenine + H2O Bacillus anthracis
-
 formate + L-kynurenine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.9 Bacillus anthracis Q81PP9 gene kynB
-
3.5.1.9 Burkholderia cenocepacia B4E9I9 gene kynB
-
3.5.1.9 Pseudomonas aeruginosa Q9I234 gene kynB
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.9 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, cleavage of the tag by tobacco etch virus, followed by another step of nickel affinity chromatography, gel filtration of the eluate Pseudomonas aeruginosa
3.5.1.9 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, cleavage of the tag by tobacco etch virus, followed by another step of nickel affinity chromatography, gel filtration of the eluate Burkholderia cenocepacia
3.5.1.9 recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, cleavage of the tag by tobacco etch virus, followed by another step of nickel affinity chromatography, gel filtration of the eluate Bacillus anthracis

Reaction

EC Number Reaction Comment Organism Reaction ID
3.5.1.9 N-formyl-L-kynurenine + H2O = formate + L-kynurenine the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism, proposed mechanism with intermediate state, overview Pseudomonas aeruginosa
a
3.5.1.9 N-formyl-L-kynurenine + H2O = formate + L-kynurenine the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism, proposed mechanism with intermediate state, overview Burkholderia cenocepacia
a
3.5.1.9 N-formyl-L-kynurenine + H2O = formate + L-kynurenine the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism, proposed mechanism with intermediate state, overview Bacillus anthracis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.9 N-formyl-L-kynurenine + H2O
-
 Pseudomonas aeruginosa formate + L-kynurenine
-
 ?
3.5.1.9 N-formyl-L-kynurenine + H2O
-
 Burkholderia cenocepacia formate + L-kynurenine
-
 ?
3.5.1.9 N-formyl-L-kynurenine + H2O
-
 Bacillus anthracis formate + L-kynurenine
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.9 dimer 2 * 40000, about, recombinant enzyme, SDS-PAGE Pseudomonas aeruginosa
3.5.1.9 dimer 2 * 40000, about, recombinant enzyme, SDS-PAGE Burkholderia cenocepacia
3.5.1.9 dimer 2 * 40000, about, recombinant enzyme, SDS-PAGE Bacillus anthracis

Synonyms

EC Number Synonyms Comment Organism
3.5.1.9 KynB
-
 Pseudomonas aeruginosa
3.5.1.9 KynB
-
 Burkholderia cenocepacia
3.5.1.9 KynB
-
 Bacillus anthracis
3.5.1.9 kynurenine formamidase
-
 Pseudomonas aeruginosa
3.5.1.9 kynurenine formamidase
-
 Burkholderia cenocepacia
3.5.1.9 kynurenine formamidase
-
 Bacillus anthracis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.9 25
-
 assay at Pseudomonas aeruginosa
3.5.1.9 25
-
 assay at Burkholderia cenocepacia
3.5.1.9 25
-
 assay at Bacillus anthracis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.9 43.94
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Burkholderia cenocepacia
3.5.1.9 50.56
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Bacillus anthracis
3.5.1.9 114.2
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.9 7.4
-
 assay at Pseudomonas aeruginosa
3.5.1.9 7.4
-
 assay at Burkholderia cenocepacia
3.5.1.9 7.4
-
 assay at Bacillus anthracis

General Information

EC Number General Information Comment Organism
3.5.1.9 evolution the enzyme differs between eukaryotes and prokaryotes Pseudomonas aeruginosa
3.5.1.9 evolution the enzyme differs between eukaryotes and prokaryotes Burkholderia cenocepacia
3.5.1.9 evolution the enzyme differs between eukaryotes and prokaryotes Bacillus anthracis
3.5.1.9 metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Pseudomonas aeruginosa
3.5.1.9 metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Burkholderia cenocepacia
3.5.1.9 metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Bacillus anthracis
3.5.1.9 additional information active site structure analysis, overview. The enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing Pseudomonas aeruginosa
3.5.1.9 additional information active site structure analysis, overview. The enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing Burkholderia cenocepacia
3.5.1.9 additional information sequence comparisons. Active site structure analysis, overview. The enzyme contains a crowded binuclear zinc catalytic site primed to generate a potent nucleophile, the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing Bacillus anthracis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.9 77.1
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Burkholderia cenocepacia
3.5.1.9 116.5
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Pseudomonas aeruginosa
3.5.1.9 126.4
-
 N-formyl-L-kynurenine pH 7.4, 25°C, recombinant enzyme Bacillus anthracis