Literature summary for 4.1.99.1 extracted from

Phillips, R.S.; Buisman, A.A.; Choi, S.; Hussaini, A.; Wood, Z.A.
The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine implications for the reaction mechanism (2018), Acta Crystallogr. Sect. D, 74, 748-759 .

Cloned(Commentary)

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 (Commentary)

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

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

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

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 [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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A853	-	-
Proteus vulgaris	P28796	-	-

Purification (Commentary)

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

Reaction	Comment	Organism	Reaction ID
L-tryptophan + H2O = indole + pyruvate + NH3	reaction via formation of quinonoid intermediate	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

Subunits	Comment	Organism
?	x * 54000, recombinant enzyme, SDS-PAGE	Proteus vulgaris

Synonyms

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.8	-	assay at	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	dependent on	Escherichia coli
pyridoxal 5'-phosphate	dependent on	Proteus vulgaris

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
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

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 [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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