Information on EC 4.1.99.1 - tryptophanase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
4.1.99.1
-
RECOMMENDED NAME
GeneOntology No.
tryptophanase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-tryptophan + H2O = indole + pyruvate + NH3
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
beta-elimination
elimination
-
-
alpha,beta-position of amino acid; beta-position of amino acid
-
replacement
-
-
beta-position of amino acid
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Tryptophan metabolism
-
-
tryptophan metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
L-tryptophan indole-lyase (deaminating; pyruvate-forming)
A pyridoxal-phosphate protein, requiring K+. The enzyme cleaves a carbon-carbon bond, releasing indole and an unstable enamine product that tautomerizes to an imine form, which undergoes a hydrolytic deamination to form pyruvate and ammonia. The latter reaction, which can occur spontaneously, can also be catalysed by EC 3.5.99.10, 2-iminobutanoate/2-iminopropanoate deaminase. Also catalyses 2,3-elimination and beta-replacement reactions of some indole-substituted tryptophan analogues of L-cysteine, L-serine and other 3-substituted amino acids.
CAS REGISTRY NUMBER
COMMENTARY hide
9024-00-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Escherichia aurescens
-
-
-
Manually annotated by BRENDA team
B1t-7A
-
-
Manually annotated by BRENDA team
strain K1
-
-
Manually annotated by BRENDA team
strain K12
-
-
Manually annotated by BRENDA team
gene tnaA
-
-
Manually annotated by BRENDA team
strain O157:H7 Sakai
-
-
Manually annotated by BRENDA team
strain SVS 370
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in Acetobacter sp.
-
-
-
Manually annotated by BRENDA team
no activity in Achromobacter sp.
-
-
-
Manually annotated by BRENDA team
no activity in Agrobacterium sp.
-
-
-
Manually annotated by BRENDA team
no activity in Alcaligenes sp.
-
-
-
Manually annotated by BRENDA team
no activity in Azotobacter sp.
-
-
-
Manually annotated by BRENDA team
no activity in Clostridium sp.
-
-
-
Manually annotated by BRENDA team
no activity in Flavobacterium sp.
-
-
-
Manually annotated by BRENDA team
no activity in Mycoplasma sp.
-
-
-
Manually annotated by BRENDA team
no activity in Pseudomonas sp.
-
-
-
Manually annotated by BRENDA team
no activity in Rhizobium sp.
-
-
-
Manually annotated by BRENDA team
no activity in Salmonella sp.
-
-
-
Manually annotated by BRENDA team
no activity in Serratia sp.
-
-
-
Manually annotated by BRENDA team
no activity in Xanthomonas sp.
-
-
-
Manually annotated by BRENDA team
Paracolobactrum coliforme
-
-
-
Manually annotated by BRENDA team
Paracolobactrum sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain W83
-
-
Manually annotated by BRENDA team
strain W83
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Shigella alkalescens
-
-
-
Manually annotated by BRENDA team
Sphaerophorus sp.
-
-
-
Manually annotated by BRENDA team
strain K-7
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxindolyl-L-alanine + H2O
?
show the reaction diagram
-
-
-
?
4-methyl-Trp + H2O
?
show the reaction diagram
-
52% of the activity with L-Trp
-
-
-
5-hydroxy-L-Trp + H2O
?
show the reaction diagram
5-hydroxyindole + L-Cys
5-hydroxy-L-Trp + ?
show the reaction diagram
-
-
-
-
-
5-hydroxyindole + S-methyl-L-Cys
5-hydroxy-L-Trp + ?
show the reaction diagram
-
-
-
-
-
5-methyl-L-Trp + H2O
?
show the reaction diagram
5-methylindole + L-Cys
5-methyl-L-Trp + ?
show the reaction diagram
-
-
-
-
-
5-methylindole + S-methyl-L-Cys
5-methyl-L-Trp + ?
show the reaction diagram
-
-
-
-
-
6-methyl-Trp + H2O
?
show the reaction diagram
-
44% of the activity with L-Trp
-
-
-
alpha,beta-diaminopropionic acid
?
show the reaction diagram
-
-
-
-
-
beta-(benzimidazol-1-yl)-L-alanine + H2O
?
show the reaction diagram
beta-chloro-L-Ala + H2O
?
show the reaction diagram
beta-chloroalanine + H2O
?
show the reaction diagram
beta-chloroalanine + H2O
pyruvate + NH4+ + Cl-
show the reaction diagram
-
-
-
-
?
Cys + H2O
?
show the reaction diagram
Cys + indole
Trp + ?
show the reaction diagram
cysteine sulfinic acid + H2O
?
show the reaction diagram
-
-
-
-
-
D-serine
pyruvate + NH3
show the reaction diagram
-
with high diammonium hydrogen phosphate concentration, e.g. 1.2 M
-
-
?
D-serine + indole
L-tryptophan + H2O
show the reaction diagram
D-Trp + H2O
indole + pyruvate + NH4+
show the reaction diagram
in the presence of high concentrations of ammonium phosphate
-
-
?
indole + L-Cys
L-Trp + ?
show the reaction diagram
indole + L-Ser
L-Trp + ?
show the reaction diagram
-
-
-
-
indole + pyruvate + NH4+
L-Trp + H2O
show the reaction diagram
indole + S-methyl-L-Cys
L-Trp + ?
show the reaction diagram
L-Ser + H2O
pyruvate + H2O
show the reaction diagram
-
-
-
-
?
L-serine + indole
L-tryptophan + H2O
show the reaction diagram
-
-
-
-
?
L-Trp + H2O
indole + pyruvate + NH3
show the reaction diagram
-
effects of temperature and hydrostatic pressure on the equilibria and rate constants for quinoid intermediate formation from L-Trp and L-Met with H463 mutant enzyme
-
-
?
L-Trp + H2O
indole + pyruvate + NH4+
show the reaction diagram
L-Trp + H2O
pyruvate + NH3 + indole
show the reaction diagram
L-tryptophan + H2O
indole + pyruvate + NH3
show the reaction diagram
O-benzylserine + H2O
?
show the reaction diagram
-
-
-
-
-
O-methylserine + H2O
?
show the reaction diagram
-
-
-
-
-
S-(2-nitrophenyl)-L-cysteine + H2O
2-nitrobenzenethiolate + pyruvate + NH4+
show the reaction diagram
-
-
-
-
ir
S-(2-nitrophenyl)-L-cysteine + H2O
2-nitrophenol + pyruvate + NH4+
show the reaction diagram
S-(2-nitrophenyl)-L-cysteine + H2O
?
show the reaction diagram
-
-
-
-
r
S-(o-nitrophenyl)-L-cysteine
?
show the reaction diagram
-
-
-
?
S-benzyl-L-Cys + H2O
phenylmethanethiol + pyruvate + NH4+
show the reaction diagram
S-benzylcysteine + H2O
?
show the reaction diagram
S-benzylcysteine + H2O
pyruvate + NH4+ + phenylmethanethiol
show the reaction diagram
-
-
-
-
?
S-ethyl-L-Cys + H2O
ethanethiol + pyruvate + NH4+
show the reaction diagram
S-ethyl-L-cysteine + H2O
?
show the reaction diagram
S-ethyl-L-cysteine + H2O
ethanethiol + pyruvate + NH4+
show the reaction diagram
S-ethyl-L-cysteine + H2O
pyruvate + NH4+ + ethanethiol
show the reaction diagram
-
-
-
-
?
S-ethylcysteine + H2O
?
show the reaction diagram
-
-
-
-
-
S-methyl-L-Cys + H2O
?
show the reaction diagram
S-methyl-L-Cys + H2O
methanethiol + pyruvate + NH4+
show the reaction diagram
S-methyl-L-Cys + H2O
pyruvate + NH4+ + methanethiol
show the reaction diagram
-
-
-
-
?
S-methyl-L-Cys + indole
Trp + ?
show the reaction diagram
S-methyl-L-cysteine
?
show the reaction diagram
-
-
-
?
S-methyl-L-cysteine + H2O
?
show the reaction diagram
S-methyl-L-cysteine + H2O
methanethiol + pyruvate + NH4+
show the reaction diagram
S-o-nitrophenyl-L-Cys + H2O
o-nitrophenol + pyruvate + NH4+
show the reaction diagram
S-o-nitrophenyl-L-Cys + H2O
o-nitrothiophenol + pyruvate + NH4+
show the reaction diagram
-
-
-
-
?
S-o-nitrophenyl-L-Cys + indole
Trp + ?
show the reaction diagram
-
reaction readily proceeds in water, it becomes impossible in water-organic solvents
-
-
S-o-nitrophenyl-L-cysteine + H2O
o-nitrothiophenol + pyruvate + NH4+
show the reaction diagram
-
-
-
-
?
Ser + H2O
?
show the reaction diagram
Ser + indole
Trp + ?
show the reaction diagram
Ser + indole
Trp + H2O
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-Trp + H2O
indole + pyruvate + NH4+
show the reaction diagram
L-tryptophan + H2O
indole + pyruvate + NH3
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cl-
-
bound to enzyme, possibly required for stabilization of subunit interactions
Cs+
-
activates
Li+
-
activates
Mg2+
-
bound to enzyme
Na+
-
activates
NaCl
-
maximal activity for the enzyme in whole cells: 0.4-0.5 M
sulfate
two ions bound two the active site of the enzyme, one of the sulfate ions interacts with both the transferase and PLP-binding domains and appears to be responsible for holding the enzyme in its closed conformation
additional information
-
TnaA is not affected by K+ or Na+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2,3-di-O-methyl)-alpha-cyclodextrin
-
-
(2,3-di-O-methyl)-beta-cyclodextrin
-
-
(2-hydroxypropyl)-alpha-cyclodextrin
-
-
(2-hydroxypropyl)-beta-cyclodextrin
-
-
2-amino-4-(benzimidazol-1-yl)butyric acid
-
i.e. homo-BZI-Ala, a potent competitive inhibitor
2-amino-5-(benzimidazol-1-yl)pentanoic acid
-
i.e. bishomo-BZI-Ala, weak inhibition
4-phenyl-2-aminobutyrate
-
-
alpha-amino-9,10-dihydro-9,10-dioxo-2-anthracenepropanoic acid
-
noncompetitive inhibition
anthranilic acid
-
competitive
beta-(benzimidazol-1-yl)-L-alanine
-
competitive, is also a good substrate for the wild-type and mutant enzymes
beta-phenyllactate
-
-
beta-phenylserine
-
-
Bifidobacterium adolescentis SPM0212
-
inhibits the proliferation of human colon cancer cell lines and it inhibits harmful fecal enzymes of rat intestinal microflora, including alpha-glucuronidase, alpha-glucosidase, tryptophanase, and urease
-
cyclodextrin
-
mixed type inhibition, competitive and non-competitive with inhibitor constants for different cyclodextrins between 0.5 and 10 mM
-
D,L-homophenylalanine
-
-
diammonium hydrogen phosphate
-
diammonium hydrogen phosphate serves as an inhibitor of tryptophanase when L-serine is substrate, the activity decreases with increasing diammonium hydrogen phosphate
Ethionine
guanidine hydrochloride
-
-
indoleacrylate
-
-
indolepropionate
-
-
Indolepyruvate
-
-
L-alanine
-
-
L-Met
-
competitive
L-methionine
-
-
L-phenylalanine
-
-
L-Trp
L-tryptophan
-
-
L-tryptophan ethylester
-
competitive inhibition
N-acetyl-L-tryptophan
-
noncompetitive inhibition
oxindolyl-L-Ala
oxindolyl-L-alanine
-
-
profilin 1
-
substrate analog
-
S-phenylbenzoquinone-L-tryptophan
-
uncompetitive inhibition
-
[2,3,6-tri-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
[2,3-di-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
[2,3-di-O-(2'-methoxyethyl)]-beta-cyclodextrin
-
-
[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-beta-cyclodextrin
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
diammonium hydrogen phosphate
-
diammonium hydrogen phosphate serves as an activator on tryptophan synthesis from D-serine, maximum activity at 20% saturation concentration
sRNA
-
dimers of plasmid ColE1 make an sRNA that interacts directly with the enzyme and enhance its substrate affinity
-
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.7
5-hydroxy-L-Trp
-
alpha,beta-elimination
1.8
5-methyl-L-Trp
-
alpha,beta-elimination
0.6
alpha,beta-diaminopropionic acid
-
strain B/1t7-A
0.0236 - 0.315
beta-(benzimidazol-1-yl)-L-alanine
1.2 - 6.15
beta-chloroalanine
110
cysteine sulfinic acid
-
strain B/1t7-A
0.019 - 4.1
indole
1 - 2.8
L-Cys
4.64 - 160
L-Ser
0.00181 - 1.47
L-Trp
0.15 - 0.347
L-tryptophan
345
NH4Cl
-
-
0.11
O-benzylserine
-
strain B/1t7-A
4
O-methylserine
-
strain B/1t7-A
10 - 70
pyruvate
0.18
S-(o-nitrophenyl)-L-cysteine
-
pH 8.0, 25C
0.065 - 0.15
S-benzylcysteine
0.26 - 3.3
S-ethyl-L-cysteine
1
S-ethylcysteine
-
strain B/1t7-A, 25C
0.18 - 15.2
S-methyl-L-Cys
0.87 - 16
S-methyl-L-cysteine
10
S-methylcysteine
-
strain B/1t7-A, 37C
0.06 - 0.18
S-o-nitrophenyl-L-Cys
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.22 - 5.6
beta-(benzimidazol-1-yl)-L-alanine
3 - 12.4
beta-chloro-L-Ala
0.0932
beta-chloroalanine
Proteus vulgaris
-
pH 7.8, 37C, mutant enzyme Y72F
0.0303
L-Ser
Proteus vulgaris
-
pH 7.8, 37C, mutant enzyme Y72F
0.24 - 42
L-Trp
0.45 - 6.8
L-tryptophan
13.9 - 44
S-(2-nitrophenyl)-L-cysteine
1.1 - 5.2
S-benzyl-L-Cys
0.00046
S-benzylcysteine
Proteus vulgaris
-
pH 7.8, 37C, mutant enzyme Y72F
0.4 - 6
S-ethyl-L-Cys
0.035 - 1.87
S-ethyl-L-cysteine
0.0051 - 5
S-methyl-L-Cys
0.2 - 0.5
S-methyl-L-cysteine
0.423
S-o-nitrophenyl-L-Cys
Proteus vulgaris
-
pH 7.8, 37C, mutant enzyme Y72F
0.00004
Trp
Proteus vulgaris
-
pH 7.8, 37C, mutant enzyme Y72F
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.3 - 18.67
beta-(benzimidazol-1-yl)-L-alanine
194446
0.77 - 2.4
beta-chloro-L-Ala
5942
6.87
L-Trp
Porphyromonas gingivalis
-
purified recombinant tryptophanase in 200 mM potassium phosphate buffer (pH 7.5), at 37C
420
1.96 - 30
L-tryptophan
119
29 - 510
S-(2-nitrophenyl)-L-cysteine
19198
11 - 81
S-benzyl-L-Cys
4302
0.28 - 9
S-ethyl-L-Cys
3507
0.6 - 7.15
S-ethyl-L-cysteine
2417
0.06 - 0.33
S-methyl-L-Cys
1801
0.23 - 0.25
S-methyl-L-cysteine
929
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.77
(2,3-di-O-methyl)-alpha-cyclodextrin
-
-
11.94
(2,3-di-O-methyl)-beta-cyclodextrin
-
-
4.97
(2-hydroxypropyl)-alpha-cyclodextrin
-
-
2.97
(2-hydroxypropyl)-beta-cyclodextrin
-
-
0.0134
2-amino-4-(benzimidazol-1-yl)butyric acid
-
wild-type enzyme, pH 8.0, 25C
0.6
2-amino-5-(benzimidazol-1-yl)pentanoic acid
-
above, wild-type enzyme, pH 8.0, 25C
174
alpha-amino-9,10-dihydro-9,10-dioxo-2-anthracenepropanoic acid
-
in 50 mM potassium phosphate buffer (pH 7.8), at 25C
0.3
beta-(benzimidazol-1-yl)-L-alanine
-
wild-type enzyme, pH 8.0, 25C
0.067
D,L-homophenylalanine
-
pH 7.8
14
L-alanine
-
pH 7.8
10.3
L-methionine
-
pH 7.8
14.2
L-phenylalanine
-
pH 7.8
0.21
L-tryptophan
-
pH 7.8
52
L-tryptophan ethylester
-
in 50 mM potassium phosphate buffer (pH 7.8), at 25C
48
N-acetyl-L-tryptophan
-
in 50 mM potassium phosphate buffer (pH 7.8), at 25C
0.005
oxindolyl-L-alanine
-
in 50 mM potassium phosphate buffer (pH 7.8), at 25C
101
S-phenylbenzoquinone-L-tryptophan
-
in 50 mM potassium phosphate buffer (pH 7.8), at 25C
-
0.24
[2,3,6-tri-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
2.15
[2,3-di-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
0.68
[2,3-di-O-(2'-methoxyethyl)]-beta-cyclodextrin
-
-
0.52
[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-alpha-cyclodextrin
-
-
1.36
[2,3-di-O-methyl-6-O-(2'-methoxyethyl)]-beta-cyclodextrin
-
-
additional information
additional information
-
Ki-values for reaction in aqueous methanol
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.508
-
cell extract from strain MG1655H, harvested at OD600 of 0.8, at 37C
0.747
-
cell extract from strain MG1655, harvested at OD600 of 0.4, at 37C
0.774
-
cell extract from strain MG1406, harvested at OD600 of 0.4, at 37C
0.808
-
cell extract from strain MG1655H, harvested at OD600 of 0.4, at 37C
0.818
-
cell extract from strain MG1655, harvested at OD600 of 0.8, at 37C
1.475
-
cell extract from strain MG1406, harvested at OD600 of 0.8, at 37C
9.24
-
-
10
substrate S-(o-nitrophenyl)-L-cysteine, pH 8.0, 25C
34
-
substrate S-(o-nitrophenyl)-L-cysteine, pH 8.0, 25C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2 - 8.8
-
strain B/1t7-A
7.5 - 9
-
immobilized enzyme
7.5
-
assay at
7.5 - 8.4
-
-
8 - 10
-
soluble enzyme
8.5
-
degradation of Trp
8.5 - 9.5
-
synthesis of L-Trp from indole and L-Cys or S-methyl-L-Cys
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9.5
-
pH dependence of quinonoid intermediate formation, overview
7 - 9.8
-
pH 7: about 45% of maximal activity, pH 9.8: about 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
assay at room temperature
25
-
assay at
30
with S-(2q-nitrophenyl)-L-cysteine
additional information
-
above 50C, with S-(2-nitrophenyl)-L-cysteine
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2
calculated from the deduced amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
-
4 * 46000, SDS-PAGE
51000
2 * 51000, SDS-PAGE, gel filtration
52800
-
4 * 52800
54000
-
4 * 54000, SDS-PAGE
57000
-
4 * 57000, equilibrium sedimentation in 6 M guanidine hydrochloride
107800
gel filtration
210000
216000
-
aggregation to form species with MW 432000 Da, 648000 Da, and 864000 Da, sucrose density gradient centrifugation, equilibrium sedimentation
220000
222000
-
ultracentrifugation and sedimentation measurement
223000
-
strain K-12, equilibrium sedimentation
244000
-
equilibrium sedimentation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo-form of enzyme without bound pyridoxal 5-phosphate but with two bound sulfate ions, hanging drop vapor diffusion method; strong crystal contacts occur on the flat surface of the protein and that the size of crystal contact surface seems to correlate with the diffraction quality of the crystal. The tryptophanase structure, solved in its apo form, does not have covalent PLP bound in the active site, but two sulfate ions. The sulfate ions occupy the phosphoryl-binding site of PLP and the binding site of the alpha-carboxyl of the natural substrate tryptophan. One of the sulfate ions makes extensive interactions with both the transferase and PLP-binding domains of the protein and appears to be responsible for holding the enzyme in its closed conformation
apo-form of the enzyme, hanging drop vapor diffusion method
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crystallized in the apo form by the hanging-drop vapour-diffusion method using polyethylene glycol 400 as a precipitant and magnesium chloride as an additive. The crystals belong to the orthorhombic space group F222, with unit-cell parameters a = 118.4 A, b = 120.1 A, c = 171.2 A. Contains a monomer in the asymmetric unit with a solvent content of 55%. Tryptophanase mutants W330F and Y74F are crystallized under the same conditions and the crystals diffracted to a resolution limit of 1.9 A
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mutant enzymes Y74F and C298S, hanging drop vapor diffusion method, using 30% (w/v) PEG 400, 100 mM HEPES pH 7.5, 200 mM MgCl2, 5 mM 2-mercaptoethanol
in complex with oxindolyl-L-alanine and with L-tryptophan and L-serine
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 10
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25C, 28 h, stable
37363
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
-
stable in presence of K+, gradual loss of activity in absence of K+
40 - 70
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the enzyme is stable up to 40C, but only 40% of the activity remains at 60C, the enzyme is completely inactivated at temperatures above 70C
63
-
10 min, holoenzyme, about 45% loss of activity
80
-
5 min, Tna1 retains alomost complete activity, Tna2 is inactivated
90
-
5 min, Tna1 retains more than 70% of activity, Tna2 is inactivated
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity decreases at pressures above 50 MPa, and by 100 MPa is less than 10% of the activity at 1 bar, initial increase in activity with pressure, reaching a maximum of about 140% at 30 MPa.
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greater than 90% activity remaining at 100 MPa, initial increase in activity with pressure, reaching a maximum of about 140% at 60 MPa.
immobilized enzyme shows higher thermal stability and resistance to a denaturing agent such as guanidine-HCl than the soluble enzyme
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rapid inactivation by visible light irradiation in presence of pyridoxal 5'-phosphate. Photoinactivation follows pseudo-first-order kinetics
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repeated slow freezing at -20C and subsequent thawing at room temperature causes the enzyme to aggregate
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when used repeatedly in a batch system or continously in a flow system in the absence of added pyridoxal 5'-phosphate, immobilized holo-tryptophanase gradually loses its original activity, pyridoxal 5'-phosphate restores its initial activity
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 8-10 mg/ml in 0.1 M potassium phosphate buffer, pH 7.8, containing 0.1 mM pyridoxal phosphate and 1.0 mM dithiothreitol, stable for up to 1 year
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-70C, stored in 0.1 M potassium phosphate buffer, pH 6.8
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5C, stored as a suspension in 0.1 M potassium phosphate buffer, pH 8.0, containing 20% v/v glycerol, 0.1 mM pyridoxal 5'-phosphate, 10 mM mercaptoethanol and 60% saturated ammonium sulfate, stable for more than 5 months
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stored as a suspension under nitrogen in 0.1 M potassium phosphate, pH 8.0, 0.2 mM dithiothreitol, 1 mM EDTA, and 2 M (NH4)2SO4
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
commercial product
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DEAE-Sephadex A-50 gel filtration or DEAE-Sepharose 4B column chromatography, and Sephacryl S-300 gel filtration
glutathione-Sepharose column chromatography
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hydrophobic interaction chromatography (phenyl-Sepharose)
hydrophobic interaction chromatography (Sepharose CL-aB)
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mutant enzyme H463F
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no added pyridoxal 5-phosphate during purification procedure resulting in the inactive apo-form of the enzyme
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed as glutathione S-transferase fusion protein
expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3) tn5:tnaA
expressed in wine yeast strains Saccharomyces cerevisiae strains YHUM272a and VIN13, expression of enzyme results in a strong increase of passion fruit aroma in wine
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expression in Escherichia coli
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gene tnaA
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gene tnaA, chromosomal TnaA-sfGFP in Escherichia coli strain GL69
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gene tnaA, expression in Escherichia coli stain BL21(DE3)
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the endogenous oxidative stress in ibpAB cells leads to increased expression of tryptophanase
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the tryptophase operon expression is induced by L-tryptophan
tryptophanase activity is decreased by 43% after lactic acid bacteria treatment (300 billion CFU/g twice a day for 2 weeks)
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tryptophanase is upregulated in the noninvasive ibeR deletion mutant BR2
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C298S
the mutant displays reduced activity, subsequent to incubation at 2C, the mutant Trpase loses about 90% of its activity
Y74F
the mutant displays reduced activity, the Y74F mutant has low activity at 25C and its residual activity is further reduced by cooling
H463F
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site-directed mutagenesis, the mutant shows very low activity for elimination of indole but is still competent to form a quinonoid intermediate from L-tryptophan, it shows high activity with substrate beta-(benzimidazol-1-yl)-L-alanine
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C298S
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the mutant displays reduced activity, subsequent to incubation at 2C, the mutant Trpase loses about 90% of its activity
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W330F
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the mutant displays reduced activity, subsequent to incubation at 2C, the mutant Trpase loses about 90% of its activity
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Y74F
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the mutant displays reduced activity, the Y74F mutant has low activity at 25C and its residual activity is further reduced by cooling
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Y72F
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mutation leads to a decrease in activity for L-tryptophan by 50000fold and to a considerable rearrangement of the active site. This rearrangement leads to an increase of room around the alpha -C atom of any bound amino acid, such that covalent binding of alpha -methyl-substituted amino acids becomes possible (which cannot be realized in wild-type Trpase)
M383I
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Tna2, slight increase of catalytic activity
S395G
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Tna2, slight increase of catalytic activity
V382M
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Tna2, slight reduction of catalytic activity
V382M/M383I/S395G
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Tna2, 2fold increase in catalytic activity
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
renaturation after exposure to 8 M urea
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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encapsulation of enzyme in wet nanoporous silica gels to selectively stabilize tertiary and quarternary protein conformations and to develop bioreactors and biosensors
drug development
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enzyme might serve as a target for antibiotics
food industry
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expression of enzyme in wine yeast results in a strong increase of passion fruit aroma in wine
medicine
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transposon insertion in gene results in a strain unable to form biofilm on polystyrene and to adhere to human pneumocyte cells
synthesis
production of L-tryptophan and related amino acids
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