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Literature summary for 3.5.1.9 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
Search for more literature for 3.5.1.9

Cloned(Commentary)

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

Crystallization (Comment) Organism
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
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
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

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

KM Value [mM]

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

Metals/Ions

Metals/Ions Comment Organism Structure
Cd2+ active site-bound Burkholderia cenocepacia
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
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
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
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
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
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]

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

Purification (Comment) Organism
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
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
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

Reaction Comment Organism Reaction ID
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
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
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)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information Comment Organism
evolution the enzyme differs between eukaryotes and prokaryotes Pseudomonas aeruginosa
evolution the enzyme differs between eukaryotes and prokaryotes Burkholderia cenocepacia
evolution the enzyme differs between eukaryotes and prokaryotes Bacillus anthracis
metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Pseudomonas aeruginosa
metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Burkholderia cenocepacia
metabolism the second stage of tryptophan catabolism is catalysed by kynurenine formamidase Bacillus anthracis
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
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
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]

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