The phosphate of pyridoxal-5'-phosphate is an acid/base catalyst in the mechanism of Pseudomonas fluorescens kynureninase
Phillips, R.S.; Scott, I.; Paulose, R.; Patel, A.; Barron, T.C.; FEBS J. 281, 1100-1109 (2014) 
Data extracted from this reference:
Cloned(Commentary)
EC Number
Cloned (Commentary)
Organism
3.7.1.3
expression of the recombinant enzyme in Escherichia coli strain DH5alpha
Pseudomonas fluorescens
Engineering
EC Number
Protein Variants
Commentary
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
Commentary
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
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
3.7.1.3
L-kynurenine + H2O
Pseudomonas fluorescens
-
anthranilate + L-alanine
-
-
?
Organism
EC Number
Organism
UniProt
Commentary
Textmining
3.7.1.3
Pseudomonas fluorescens
P83788
gene kynU
-
Purification (Commentary)
EC Number
Purification (Commentary)
Organism
3.7.1.3
recombinant enzyme from Escherichia coli strain DH5alpha
Pseudomonas fluorescens
Reaction
EC Number
Reaction
Commentary
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
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
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
733832
Pseudomonas fluorescens
?
-
-
-
?
3.7.1.3
L-kynurenine + H2O
-
733832
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
733832
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
733832
Pseudomonas fluorescens
benzoate + pyruvate + ammonium
-
-
-
?
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
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
Commentary
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
Commentary
Organism
3.7.1.3
8
-
assay at
Pseudomonas fluorescens
Cofactor
EC Number
Cofactor
Commentary
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
Commentary
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
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.7.1.3
expression of the recombinant enzyme in Escherichia coli strain DH5alpha
Pseudomonas fluorescens
Cofactor (protein specific)
EC Number
Cofactor
Commentary
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
Engineering (protein specific)
EC Number
Protein Variants
Commentary
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 (protein specific)
EC Number
Inhibitors
Commentary
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
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
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
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ID
3.7.1.3
L-kynurenine + H2O
Pseudomonas fluorescens
-
anthranilate + L-alanine
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.7.1.3
recombinant enzyme from Escherichia coli strain DH5alpha
Pseudomonas fluorescens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
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
733832
Pseudomonas fluorescens
?
-
-
-
?
3.7.1.3
L-kynurenine + H2O
-
733832
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
733832
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
733832
Pseudomonas fluorescens
benzoate + pyruvate + ammonium
-
-
-
?
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.7.1.3
37
-
assay at
Pseudomonas fluorescens
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
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 (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.7.1.3
8
-
assay at
Pseudomonas fluorescens
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
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
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
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