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Literature summary extracted from

  • Phillips, R.S.; Scott, I.; Paulose, R.; Patel, A.; Barron, T.C.
    The phosphate of pyridoxal-5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase (2014), FEBS J., 281, 1100-1109.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.7.1.3 expression of the recombinant enzyme in Escherichia coli strain DH5alpha Pseudomonas fluorescens

Protein Variants

EC Number Protein Variants Comment Organism
3.7.1.3 Y226F site-directed mutagenesis, the mutant has approximately 3000fold lower activity than wild-type, and does not show the pKa values of 6.8 on kcat and 6.5 and 8.8 on kcat/Km seen for the wild-type enzyme Pseudomonas fluorescens

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.7.1.3 (alphaS,4S)-dihydrokynurenine the (alphaS,4R) diastereomer is a slow substrate for a retro-aldol reaction, and the (alphaS,4S) isomer is a potent competitive inhibitor, proposed mechanism for reaction of dihydrokynurenine Pseudomonas fluorescens
3.7.1.3 3-hydroxyhippuric acid a competitive inhibitor Pseudomonas fluorescens
3.7.1.3 5-bromodihydrokynurenine a potent transition-state analogue inhibitor Pseudomonas fluorescens
3.7.1.3 S-(2-aminophenyl)-L-cysteine dioxide
-
 Pseudomonas fluorescens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.7.1.3 L-kynurenine + H2O Pseudomonas fluorescens
-
 anthranilate + L-alanine
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.7.1.3 Pseudomonas fluorescens P83788 gene kynU
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.7.1.3 recombinant enzyme from Escherichia coli strain DH5alpha Pseudomonas fluorescens

Reaction

EC Number Reaction Comment Organism Reaction ID
3.7.1.3 L-kynurenine + H2O = anthranilate + L-alanine the proposed mechanism of the retro-Claisen reaction requires extensive acid/base catalysis, the phosphate of pyridoxal 5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase, Tyr226 hydrogen bonds to the phosphate of the pyridoxal 5'-phosphate cofactor. Tyr226 mediates proton transfer between the substrate and the phosphate, which accelerates formation of external aldimine and gem-diol intermediates, catalytic reaction mechanism via several intermediates, e.g. external aldimine, quinonoid, ketimine, Gem-diolate, and enamine, overview Pseudomonas fluorescens
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.7.1.3 (alphaS,4R)-dihydrokynurenine + H2O the (alphaS,4R) diastereomer is a slow substrate for a retro-aldol reaction, and the (alphaS,4S) isomer is a potent competitive inhibitor, proposed mechanism for reaction of dihydrokynurenine Pseudomonas fluorescens ?
-
 ?
3.7.1.3 L-kynurenine + H2O
-
 Pseudomonas fluorescens anthranilate + L-alanine
-
 ?
3.7.1.3 L-kynurenine + H2O the enzyme catalyzes the hydrolytic Cbeta-Cgamma cleavage of L-kynurenine to alanine and anthranilic acid catalyzing a retro-Claisen reaction Pseudomonas fluorescens anthranilate + L-alanine
-
 ?
3.7.1.3 O-benzoyl-L-serine + H2O O-benzoyl-L-serine is an alternate substrate for kynureninase, which undergoes a normal beta-elimination reaction, proposed mechanism for reaction of O-benzoyl-L-serine, overview Pseudomonas fluorescens benzoate + pyruvate + ammonium
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.7.1.3 37
-
 assay at Pseudomonas fluorescens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.7.1.3 0.0058
-
 L-kynurenine enzyme mutant Y226F, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 0.1
-
 O-benzoyl-L-serine enzyme mutant Y226F, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 2.5
-
 O-benzoyl-L-serine wild-type enzyme, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 16
-
 L-kynurenine wild-type enzyme, pH 8.0, 37°C Pseudomonas fluorescens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.7.1.3 8
-
 assay at Pseudomonas fluorescens

Cofactor

EC Number Cofactor Comment Organism Structure
3.7.1.3 pyridoxal 5'-phosphate the phosphate of pyridoxal 5'-phosphate is an acid/base catalyst in the mechanism of kynureninase. Tyr226 hydrogen bonds to the phosphate of the pyridoxal 5'-phosphate cofactor Pseudomonas fluorescens

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
3.7.1.3 0.00003
-
 S-(2-aminophenyl)-L-cysteine dioxide wild-type enzyme, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 0.00092
-
 S-(2-aminophenyl)-L-cysteine dioxide enzyme mutant Y226F, pH 8.0, 37°C Pseudomonas fluorescens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.7.1.3 0.0041
-
 O-benzoyl-L-serine enzyme mutant Y226F, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 0.53
-
 L-kynurenine enzyme mutant Y226F, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 11
-
 O-benzoyl-L-serine wild-type enzyme, pH 8.0, 37°C Pseudomonas fluorescens
3.7.1.3 200
-
 L-kynurenine wild-type enzyme, pH 8.0, 37°C Pseudomonas fluorescens