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Information on EC 4.2.1.20 - tryptophan synthase and Organism(s) Pyrococcus furiosus and UniProt Accession Q8U093

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.20 tryptophan synthase
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.
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Pyrococcus furiosus
UNIPROT: Q8U093
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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
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TrpB1
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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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-serine + 1-C-(indol-3-yl)glycerol 3-phosphate = L-tryptophan + D-glyceraldehyde 3-phosphate + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
addition
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-
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-
SYSTEMATIC NAME
IUBMB Comments
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.
CAS REGISTRY NUMBER
COMMENTARY hide
9014-52-2
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-serine + 1-C-(indol-3-yl)glycerol 3-phosphate
L-tryptophan + D-glyceraldehyde 3-phosphate + H2O
show the reaction diagram
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-
-
?
L-serine + 1H-indole-5-carbaldehyde
5-formyl-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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-
?
L-serine + 5-bromoindole
5-bromo-L-tryptophan + H2O
show the reaction diagram
L-serine + 5-chloroindole
5-chloro-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
-
-
?
L-serine + 5-hydroxyindole
5-hydroxy-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
-
-
?
L-serine + 5-methoxyindole
5-methoxy-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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-
?
L-serine + 5-methylindole
5-methyl-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
-
-
?
L-serine + 5-nitroindole
5-nitro-L-tryptophan + H2O
show the reaction diagram
substrate of mutant P12L/E17G/I68V/F274S/T292S/T321A
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-
?
L-serine + indole
L-tryptophan + H2O
show the reaction diagram
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-
-
?
L-threonine + 1-C-(indol-3-yl)glycerol 3-phosphate
(2S,3S)-beta-methyltryptophan + D-glyceraldehyde 3-phosphate + H2O
show the reaction diagram
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-
-
?
L-threonine + 2-methylindole
2-methyl-(2S,3S)-beta-methyltryptophan + H2O
show the reaction diagram
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-
-
?
L-threonine + 4-fluoroindole
4-fluoro-(2S,3S)-beta-methyltryptophan + H2O
show the reaction diagram
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
show the reaction diagram
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
show the reaction diagram
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
show the reaction diagram
reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A
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-
?
L-threonine + indole
(2S,3S)-beta-methyltryptophan + H2O
show the reaction diagram
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-
-
?
1-(indol-3-yl)glycerol 3-phosphate
D-glyceraldehyde 3-phosphate + indole
show the reaction diagram
L-serine + indole
L-tryptophan + H2O
show the reaction diagram
additional information
?
-
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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?
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.02 - 1.4
1-C-(indol-3-yl)glycerol 3-phosphate
0.009 - 0.077
indole
0.6
L-serine
pH 8.0, temperature not specified in the publication
1.3
L-threonine
pH 8.0, temperature not specified in the publication
8.7 - 77
indole
0.6 - 1.2
L-serine
additional information
additional information
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thermodynamic parameters of subunit association, kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.31 - 2.9
indole
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 - 2.9
indole
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.46 - 50
1-C-(indol-3-yl)glycerol 3-phosphate
4 - 30
indole
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
4 - 330
indole
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
42500
84000 - 88000
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beta-subunit dimer, ultracentrifugation sedimentation equilibrium
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
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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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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
9fold increase in kcat with an equivalent decrease in KM
E17G/I68V/F274S/T292S/T321A
mutation in subunit TrpB1, 50fold increase of catalytic efficiency of isolated beta subunit
P12L/E17G/I68V/F274S/T292S/T321A
mutation in subunit TrpB1, 80fold increase of catalytic efficiency of isolated beta subunit
T292S
mutation in subunit TrpB1, 20fold increase of catalytic efficiency of isolated beta subunit
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant alpha-subunit from Escherichia coli
recombinant alpha-subunit, beta-subunit, and enzyme complex expressed in Escherichia coli
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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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overexpression of the alpha-subunit in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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)
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team