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EC Tree
IUBMB Comments A pyridoxal-phosphate protein. The alpha-subunit catalyses the conversion of 1-C-(indol-3-yl)glycerol 3-phosphate to indole and D-glyceraldehyde 3-phosphate (this reaction was included formerly under EC 4.1.2.8). The indole migrates to the beta-subunit where, in the presence of pyridoxal 5'-phosphate, it is combined with L-serine to form L-tryptophan. In some organisms this enzyme is part of a multifunctional protein that also includes one or more of the enzymes EC 2.4.2.18 (anthranilate phosphoribosyltransferase), EC 4.1.1.48 (indole-3-glycerol-phosphate synthase), EC 4.1.3.27 (anthranilate synthase) and EC 5.3.1.24 (phosphoribosylanthranilate isomerase). In thermophilic organisms, where the high temperature enhances diffusion and causes the loss of indole, a protein similar to the beta subunit can be found (EC 4.2.1.122). That enzyme cannot combine with the alpha unit of EC 4.2.1.20 to form a complex.
The taxonomic range for the selected organisms is: Pyrococcus furiosus The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
trps, tryptophan synthase, tryptophan synthetase, tsase, alphats, trpb2, tryptophan synthase beta, trp synthase, trpb1, beta subunit of tryptophan synthase,
more
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indoleglycerol phosphate aldolase
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L-tryptophan synthetase
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synthase, tryptophan
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tryptophan desmolase
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tryptophan synthetase
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TrpB1
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L-serine + 1-C-(indol-3-yl)glycerol 3-phosphate = L-tryptophan + D-glyceraldehyde 3-phosphate + H2O
also catalyses the conversion of serine and indole into tryptophan and water, and of indoleglycerol phosphate into indole and glyceraldehyde phosphate (the latter reaction was listed formerly as EC 4.2.1.8)
L-serine + 1-C-(indol-3-yl)glycerol 3-phosphate = L-tryptophan + D-glyceraldehyde 3-phosphate + H2O
also catalyses the conversion of serine and indole into tryptophan and water, and of indoleglycerol phosphate into indole and glyceraldehyde phosphate (the latter reaction was listed formerly as EC 4.2.1.8)
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L-serine hydro-lyase [adding 1-C-(indol-3-yl)glycerol 3-phosphate; L-tryptophan and glyceraldehyde-3-phosphate-forming]
A pyridoxal-phosphate protein. The alpha-subunit catalyses the conversion of 1-C-(indol-3-yl)glycerol 3-phosphate to indole and D-glyceraldehyde 3-phosphate (this reaction was included formerly under EC 4.1.2.8). The indole migrates to the beta-subunit where, in the presence of pyridoxal 5'-phosphate, it is combined with L-serine to form L-tryptophan. In some organisms this enzyme is part of a multifunctional protein that also includes one or more of the enzymes EC 2.4.2.18 (anthranilate phosphoribosyltransferase), EC 4.1.1.48 (indole-3-glycerol-phosphate synthase), EC 4.1.3.27 (anthranilate synthase) and EC 5.3.1.24 (phosphoribosylanthranilate isomerase). In thermophilic organisms, where the high temperature enhances diffusion and causes the loss of indole, a protein similar to the beta subunit can be found (EC 4.2.1.122). That enzyme cannot combine with the alpha unit of EC 4.2.1.20 to form a complex.
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1-(indol-3-yl)glycerol 3-phosphate
D-glyceraldehyde 3-phosphate + indole
alpha-subunit of the bienzyme complex, alpha-reaction
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?
L-serine + indole
L-tryptophan + H2O
beta-subunit of the bienzyme complex, beta-reaction
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?
1-(indol-3-yl)glycerol 3-phosphate
D-glyceraldehyde 3-phosphate + indole
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alpha-subunit of the bienzyme complex, alpha-reaction
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?
L-serine + 1-C-(indol-3-yl)glycerol 3-phosphate
L-tryptophan + D-glyceraldehyde 3-phosphate + H2O
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?
L-serine + 1H-indole-5-carbaldehyde
5-formyl-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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?
L-serine + 5-bromoindole
5-bromo-L-tryptophan + H2O
L-serine + 5-chloroindole
5-chloro-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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-
?
L-serine + 5-hydroxyindole
5-hydroxy-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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-
?
L-serine + 5-methoxyindole
5-methoxy-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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?
L-serine + 5-methylindole
5-methyl-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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?
L-serine + 5-nitroindole
5-nitro-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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?
L-serine + indole
L-tryptophan + H2O
L-threonine + 1-C-(indol-3-yl)glycerol 3-phosphate
(2S,3S)-beta-methyltryptophan + D-glyceraldehyde 3-phosphate + H2O
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?
L-threonine + 2-methylindole
2-methyl-(2S,3S)-beta-methyltryptophan + H2O
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?
L-threonine + 4-fluoroindole
4-fluoro-(2S,3S)-beta-methyltryptophan + H2O
reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
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?
L-threonine + 5-fluoroindole
5-fluoro-(2S,3S)-beta-methyltryptophan + H2O
reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
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?
L-threonine + 6-methylindole
6-methyl-(2S,3S)-beta-methyltryptophan + H2O
reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
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?
L-threonine + 7-azaindole
7-aza-(2S,3S)-beta-methyltryptophan + H2O
reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
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L-threonine + indole
(2S,3S)-beta-methyltryptophan + H2O
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?
additional information
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native tryptophan synthase can also catalyze a productive reaction with L-threonine, leading to (2S,3S)-beta-methyltryptophan. Substitution occurs in vitro with a 3.4fold higher catalytic efficiency for Ser over Thr using saturating indole. Threonine binds efficiently but decreases the affinity for indole and disrupts the allosteric signaling that regulates the catalytic cycle
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L-serine + 5-bromoindole
5-bromo-L-tryptophan + H2O
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?
L-serine + 5-bromoindole
5-bromo-L-tryptophan + H2O
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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?
L-serine + indole
L-tryptophan + H2O
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L-serine + indole
L-tryptophan + H2O
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L-serine + indole
L-tryptophan + H2O
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beta-subunit of the bienzyme complex, beta-reaction
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?
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0.02 - 1.4
1-C-(indol-3-yl)glycerol 3-phosphate
1.3
L-threonine
pH 8.0, temperature not specified in the publication
additional information
additional information
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thermodynamic parameters of subunit association, kinetics
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0.02
1-C-(indol-3-yl)glycerol 3-phosphate
cosubstrate L-serine, pH 8.0, temperature not specified in the publication
1.4
1-C-(indol-3-yl)glycerol 3-phosphate
cosubstrate L-threonine, pH 8.0, temperature not specified in the publication
0.009
indole
mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
0.042
indole
mutant M144T/N166D, pH 8.0, 75°C
0.077
indole
wild-type, pH 8.0, 75°C
8.7
indole
subunit TrpB1, mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
11
indole
subunit TrpB1, mutant E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
14
indole
subunit TrpB1, mutant T292S, pH 8.0, 75°C
20
indole
wild-type holoenzyme, pH 8.0, 75°C
77
indole
subunit TrpB1, wild-type, pH 8.0, 75°C
0.6
L-serine
pH 8.0, temperature not specified in the publication
0.6
L-serine
wild-type holoenzyme, pH 8.0, 75°C
0.7
L-serine
subunit TrpB1, mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
0.84
L-serine
subunit TrpB1, mutant T292S, pH 8.0, 75°C
1.2
L-serine
subunit TrpB1, mutant E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
1.2
L-serine
subunit TrpB1, wild-type, pH 8.0, 75°C
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1
L-serine
pH 8.0, temperature not specified in the publication
0.61
L-threonine
pH 8.0, temperature not specified in the publication
0.31
indole
subunit TrpB1, wild-type, pH 8.0, 75°C
0.31
indole
wild-type, pH 8.0, 75°C
0.83
indole
mutant M144T/N166D, pH 8.0, 75°C
1
indole
wild-type holoenzyme, pH 8.0, 75°C
1.1
indole
subunit TrpB1, mutant T292S, pH 8.0, 75°C
2.2
indole
subunit TrpB1, mutant E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
2.9
indole
mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
2.9
indole
subunit TrpB1, mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
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0.46 - 50
1-C-(indol-3-yl)glycerol 3-phosphate
1.6
L-serine
pH 8.0, temperature not specified in the publication
0.47
L-threonine
pH 8.0, temperature not specified in the publication
0.46
1-C-(indol-3-yl)glycerol 3-phosphate
cosubstrate L-threonine, pH 8.0, temperature not specified in the publication
50
1-C-(indol-3-yl)glycerol 3-phosphate
cosubstrate L-serine, pH 8.0, temperature not specified in the publication
4
indole
subunit TrpB1, wild-type, pH 8.0, 75°C
4
indole
wild-type, pH 8.0, 75°C
20
indole
mutant M144T/N166D, pH 8.0, 75°C
30
indole
mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
50
indole
wild-type holoenzyme, pH 8.0, 75°C
78
indole
subunit TrpB1, mutant T292S, pH 8.0, 75°C
200
indole
subunit TrpB1, mutant E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
330
indole
subunit TrpB1, mutant P12L/E17G/I68V/F274S/T292S/T321A, pH 8.0, 75°C
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Uniprot
brenda
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physiological function
as part of its native alphabetabetaalpha complex, subunit TrpB efficiently produces tryptophan and tryptophan analogs. Activity drops considerably when it is used as a stand-alone catalyst without the alpha-subunit. This lost activity can be recovered by mutations that reproduce the effects of complexation with the alpha-subunit
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140000
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enzyme complex alpha2beta2, ultracentrifugation sedimentation equilibrium
27500
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alpha-subunit monomer, ultracentrifugation sedimentation equilibrium
275000
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2 * 275000 + 2 * 42500, recombinant alpha2beta2 enzyme complex, SDS-PAGE
84000 - 88000
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beta-subunit dimer, ultracentrifugation sedimentation equilibrium
42500
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2 * 275000 + 2 * 42500, recombinant alpha2beta2 enzyme complex, SDS-PAGE
42500
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2 * 42500, recombinant beta-subunit, SDS-PAGE
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dimer
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2 * 42500, recombinant beta-subunit, SDS-PAGE
monomer
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1 * 27500, recombinant alpha-subunit, SDS-PAGE
tetramer
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2 * 275000 + 2 * 42500, recombinant alpha2beta2 enzyme complex, SDS-PAGE
additional information
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high association constant for the hyperthermophile enzyme subunits, pH-dependence of the molecular weights
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purified recombinant alpha-subunit, hanging drop vapour diffusion method, 10°C, reservoir solution: 0.1 M MES-NaOH, pH 6.5, 12% PEG 20000, X-ray diffraction structure determination and analysis at 2.0 A resolution
crystal structure of tryptophan synthase beta2 subunit determined at 2.2 A resolution, hanging drop vapour diffusion method. Crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 84.8 A, b = 110.5 A, c = 160.0 A
hanging drop vapour diffusion method, crystal structure of the alpha2beta2 complex, crystals belong to the orthorhombic space group of P2(1)2(1)2(1) with unit cell dimensions of a = 89.1 A, b = 220.3 A, c = 292.6 A
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wild-type in complex with L-threonine, to 1.54 A. L-threonine binds non-covalently, no formation of an electrophilic amino-acrylate intermediate. With mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A, the species is formed
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E17G/I68V/F274S/T292S/T321A
mutation in subunit TrpB1, 50fold increase of catalytic efficiency of isolated beta subunit
I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
significant increase in activity with L-threonine
M144T/N166D
5fold increase in catalytic efficiency
P12L/E17G/I68V/F274S/T292S/T321A
T292S
mutation in subunit TrpB1, 20fold increase of catalytic efficiency of isolated beta subunit
P12L/E17G/I68V/F274S/T292S/T321A
9fold increase in kcat with an equivalent decrease in KM
P12L/E17G/I68V/F274S/T292S/T321A
mutation in subunit TrpB1, 80fold increase of catalytic efficiency of isolated beta subunit
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84
1 h, 50% residual activity, mutant E17G/I68V/F274S/T292S/T321A
87
1 h, 50% residual activity, mutant P12L/E17G/I68V/F274S/T292S/T321A
95
1 h, 50% residual activity, wild-type holoenzyme, wild-type subunit TrpB1 and mutant T292S
additional information
the alpha-subunit of the enzyme has extremely high thermostability, due to increase in ion pairs, decrease in cavity volume, and entropic effects, not by hydrophobic interactions
additional information
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the alpha-subunit of the enzyme has extremely high thermostability, due to increase in ion pairs, decrease in cavity volume, and entropic effects, not by hydrophobic interactions
additional information
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inactivation temperature of the alpha-subunit is enhanced by the formation of the enzyme complex alpha2beta2 by 17°C
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recombinant alpha-subunit from Escherichia coli
recombinant alpha-subunit, beta-subunit, and enzyme complex expressed in Escherichia coli
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overexpression of the alpha-subunit in Escherichia coli
genes trpA encoding the alpha-subunit and trpB encoding the beta-subunit are coexpressed or expressed individually in Escherichia coli strain JM109
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Yamagata, Y.; Ogasahara, K.; Hioki, Y.; Lee, S.J.; Nakagawa, A.; Nakamura, H.; Ishida, M.; Kuramitsu, S.; Yutani, K.
Entropic stabilization of the tryptophan synthase alpha-subunit from a hyperthermophile, Pyrococcus furiosus. X-ray analysis and calorimetry
J. Biol. Chem.
276
11062-11071
2001
Pyrococcus furiosus (Q8U094), Pyrococcus furiosus
brenda
Ogasahara, K.; Ishida, M.; Yutani, K.
Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability
J. Biol. Chem.
278
8922-8928
2003
Pyrococcus furiosus
brenda
Lee, S.J.; Ogasahara, K.; Ma, J.; Nishio, K.; Ishida, M.; Yamagata, Y.; Tsukihara, T.; Yutani, K.
Conformational changes in the tryptophan synthase from a hyperthermophile upon alpha2beta2 complex formation: crystal structure of the complex
Biochemistry
44
11417-11427
2005
Pyrococcus furiosus
brenda
Hioki, Y.; Ogasahara, K.; Lee, S.J.; Ma, J.; Ishida, M.; Yamagata, Y.; Matsuura, Y.; Ota, M.; Ikeguchi, M.; Kuramitsu, S.; Yutani, K.
The crystal structure of the tryptophan synthase beta subunit from the hyperthermophile Pyrococcus furiosus. Investigation of stabilization factors
Eur. J. Biochem.
271
2624-2635
2004
Pyrococcus furiosus (Q8U093), Pyrococcus furiosus
brenda
Murciano-Calles, J.; Romney, D.; Brinkmann-Chen, S.; Buller, A.; Arnold, F.
A Panel of TrpB biocatalysts derived from tryptophan synthase through the transfer of mutations that mimic allosteric activation
Angew. Chem. Int. Ed. Engl.
55
11577-11581
2016
Archaeoglobus fulgidus (O28672), Escherichia coli (P0A879), Thermotoga maritima (P50909), Pyrococcus furiosus (Q8U093), Pyrococcus furiosus
brenda
Buller, A.; Van Roye, P.; Murciano-Calles, J.; Arnold, F.
tryptophan synthase uses an atypical mechanism to achieve substrate specificity
Biochemistry
55
7043-7046
2016
Pyrococcus furiosus (Q8U093), Pyrococcus furiosus ATCC 43587 (Q8U093)
brenda
Herger, M.; Van Roye, P.; Romney, D.; Brinkmann-Chen, S.; Buller, A.; Arnold, F.
Synthesis of beta-branched tryptophan analogues using an engineered subunit of tryptophan synthase
J. Am. Chem. Soc.
138
8388-8391
2016
Pyrococcus furiosus (Q8U093), Pyrococcus furiosus
brenda
Buller, A.; Brinkmann-Chen, S.; Romney, D.; Herger, M.; Murciano-Calles, J.; Arnold, F.
Directed evolution of the tryptophan synthase beta-subunit for stand-alone function recapitulates allosteric activation
Proc. Natl. Acad. Sci. USA
112
14599-14604
2015
Pyrococcus furiosus (Q8U093 and Q8U094), Pyrococcus furiosus
brenda