Cloned (Comment) | Organism |
---|---|
gene tnaA, recombinant expression of the enzyme in Escherichia coli strain BL21(DE3) | Proteus vulgaris |
gene tnaA, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Escherichia coli |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme in complex with inhibitor oxindolyl-L-alanine (OIA), hanging-drop vapor diffusion method by mixing 15 mg/ml protein with an equal volume of reservoir solution consisting of 35 mM potassium phosphate, 0.05 M HEPES, pH 7.0, 0.3 M KCl, and 11% PEG 4000, at 20°C, the crystals are transferred to reservoir solution supplemented with 20% 1:1:1 ethylene glycol:DMSO:glycerol as cryosolvent, with or without 10 mM OIA, X-ray diffraction structure determination and analysis at 2.0-2.1 A resolution | Proteus vulgaris |
Protein Variants | Comment | Organism |
---|---|---|
F464A | site-directed mutagenesis, the mutation results in a 500fold decrease in kcat/Km for L-tryptophan, with less effect on the reaction of other nonphysiological elimination substrates. The mutation has no effect on the formation of quinonoid intermediates | Escherichia coli |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
oxindolyl-L-alanine | OIA, a potent competitive inhibitor of the enzyme, transition-state analogue | Escherichia coli | |
oxindolyl-L-alanine | OIA, a potent competitive inhibitor of the enzyme, transition-state analogue. The small enzyme domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Four subunits of the tetramer of the OIA complex, the complex is clearly in the quinonoid form, modeled L-tryptophan-PLP quinonoid complex with OIP, structure overview | Proteus vulgaris |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics, stopped-flow kinetics, rate and equilibrium constants for pre-steady-state reaction of F464A Escherichia coli enzyme TIL with L-tryptophan | Escherichia coli |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-tryptophan + H2O | Escherichia coli | - |
indole + pyruvate + NH3 | - |
? | |
L-tryptophan + H2O | Proteus vulgaris | - |
indole + pyruvate + NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | P0A853 | - |
- |
Proteus vulgaris | P28796 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant enzyme from Escherichia coli strain BL21(DE3) by protamine sulfate treatment, hydrophobic interaction chromatography, and gel filtration | Proteus vulgaris |
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by protamine sulfate treatment, hydrophobic interaction chromatography, and gel filtration | Escherichia coli |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
L-tryptophan + H2O = indole + pyruvate + NH3 | reaction via formation of quinonoid intermediate | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
L-tryptophan + H2O | - |
Escherichia coli | indole + pyruvate + NH3 | - |
? | |
L-tryptophan + H2O | - |
Proteus vulgaris | indole + pyruvate + NH3 | - |
? | |
S-(2-nitrophenyl)-L-cysteine + H2O | - |
Escherichia coli | 2-nitrobenzenethiolate + pyruvate + NH3 | - |
? | |
S-(2-nitrophenyl)-L-cysteine + H2O | - |
Proteus vulgaris | 2-nitrobenzenethiolate + pyruvate + NH3 | - |
? | |
S-ethyl-L-cysteine + H2O | - |
Escherichia coli | ethanethiol + pyruvate + NH3 | - |
? | |
S-ethyl-L-cysteine + H2O | - |
Proteus vulgaris | ethanethiol + pyruvate + NH3 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 54000, recombinant enzyme, SDS-PAGE | Proteus vulgaris |
Synonyms | Comment | Organism |
---|---|---|
TIL | - |
Escherichia coli |
TIL | - |
Proteus vulgaris |
TnaA | - |
Escherichia coli |
TnaA | - |
Proteus vulgaris |
tryptophan indole-lyase | - |
Escherichia coli |
tryptophan indole-lyase | - |
Proteus vulgaris |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
22 | - |
assay at room temperature | Escherichia coli |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.009 | - |
L-tryptophan | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
0.074 | - |
S-ethyl-L-cysteine | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
2.2 | - |
S-(2-nitrophenyl)-L-cysteine | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
6 | - |
L-tryptophan | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli | |
6 | - |
S-ethyl-L-cysteine | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli | |
38 | - |
S-(2-nitrophenyl)-L-cysteine | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | dependent on | Escherichia coli | |
pyridoxal 5'-phosphate | dependent on | Proteus vulgaris |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.005 | - |
oxindolyl-L-alanine | pH and temperature not specified in the publication | Proteus vulgaris | |
0.005 | - |
oxindolyl-L-alanine | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli | |
0.0614 | - |
oxindolyl-L-alanine | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli |
General Information | Comment | Organism |
---|---|---|
evolution | residue Phe464 in Escherichia coli TIL is homologous to Phe459 in Proteus vulgaris TIL | Escherichia coli |
evolution | residue Phe464 in Escherichia coli TIL is homologous to Phe459 in Proteus vulgaris TIL | Proteus vulgaris |
additional information | the reaction intermediate quinonoid complex of pyridoxal 5'-phosphate with L-tryptophan is modeled, based on the structure with inhibitor oxindolyl-L-alanine, by replacement of the oxindole ring with indole, and then docked manually into the active site by overlaying the pyridoxal 5'-phosphate rings of OIP and the L-tryptophan-pyridoxal 5'-phosphate complex | Proteus vulgaris |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.067 | - |
L-tryptophan | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
0.672 | - |
S-ethyl-L-cysteine | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
9 | - |
S-ethyl-L-cysteine | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli | |
30 | - |
L-tryptophan | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli | |
170 | - |
S-(2-nitrophenyl)-L-cysteine | recombinant mutant F464A, pH 7.8, 22°C | Escherichia coli | |
500 | - |
S-(2-nitrophenyl)-L-cysteine | recombinant wild-type enzyme, pH 7.8, 22°C | Escherichia coli |