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Information on EC 3.1.2.29 - fluoroacetyl-CoA thioesterase
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3.1.2.29 fluoroacetyl-CoA thioesteraseIUBMB Comments
Fluoroacetate is extremely toxic. It reacts with CoA to form fluoroacetyl-CoA, which substitutes for acetyl CoA and reacts with EC 2.3.3.1 (citrate synthase) to produce fluorocitrate, a metabolite of which binds very tightly to EC 4.2.1.3 (aconitase) and halts the TCA cycle. This enzyme hydrolyses fluoroacetyl-CoA before it can react with citrate synthase, and thus confers fluoroacetate resistance on the organisms that produce it. It has been described in the poisonous plant Dichapetalum cymosum and the bacterium Streptomyces cattleya, both of which are fluoroacetate producers.
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The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
fluoroacetyl-coa thioesterase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
FlK
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
fluoroacetyl-CoA + H2O = fluoroacetate + CoA
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SYSTEMATIC NAME
IUBMB Comments
fluoroacetyl-CoA hydrolase
Fluoroacetate is extremely toxic. It reacts with CoA to form fluoroacetyl-CoA, which substitutes for acetyl CoA and reacts with EC 2.3.3.1 (citrate synthase) to produce fluorocitrate, a metabolite of which binds very tightly to EC 4.2.1.3 (aconitase) and halts the TCA cycle. This enzyme hydrolyses fluoroacetyl-CoA before it can react with citrate synthase, and thus confers fluoroacetate resistance on the organisms that produce it. It has been described in the poisonous plant Dichapetalum cymosum and the bacterium Streptomyces cattleya, both of which are fluoroacetate producers.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
fluoroacetyl-CoA + H2O
fluoroacetate + CoA
does not accept acetyl-CoA as a substrate
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?
fluoroacetyl-CoA + H2O
fluoroacetate + CoA
the enzyme is strongly selective for fluoroacetyl-CoA compared with acetyl-CoA
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additional information
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Thr 42, Glu 50, and His 76 are key catalytic residues. FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding. Fluorinated substrates bind in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. Water plays a critical role in fluorine specificity. The Phe 36 gate residue functions to exclude water from the active site
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additional information
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chemical, kinetic, and structural mechanism for the selectivity for fluoroacetyl-CoA. Following substrate binding, fluoroacetyl-CoA reacts with Glu50 to acylate this side chain. After enzyme acylation, a proton is abstracted by His 76 to generate an enolate that can break down through a proposed ketene intermediate. The pro-R proton of fluoroacetyl-CoA is specifically abstracted. The selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity
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additional information
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chemical, kinetic, and structural mechanism for the selectivity for fluoroacetyl-CoA. Following substrate binding, fluoroacetyl-CoA reacts with Glu50 to acylate this side chain. After enzyme acylation, a proton is abstracted by His 76 to generate an enolate that can break down through a proposed ketene intermediate. The pro-R proton of fluoroacetyl-CoA is specifically abstracted. The selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity
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additional information
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the high selectivity of fluoroacetyl-CoA thioesterase is achieved through catalysis rather than molecular recognition. Deprotonation at the Calpha position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10000fold compared with the nonfluorinated congene. An enzyme-anhydride intermediate is formed for the nonfluorinated substrate
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additional information
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the high selectivity of fluoroacetyl-CoA thioesterase is achieved through catalysis rather than molecular recognition. Deprotonation at the Calpha position to form a putative ketene intermediate only occurs on the fluorinated substrate, thereby accelerating the rate of hydrolysis 10000fold compared with the nonfluorinated congene. An enzyme-anhydride intermediate is formed for the nonfluorinated substrate
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?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
fluoroacetyl-CoA
substrate inhibition of mutants T42S and E50A above 0.012 and 0.019 mM, respectively; T42S mutant protein: substrate inhibition observed above 0.012 mM, E50A mutant protein: substrate inhibition observed above 0.019 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.015
fluoroacetyl-CoA
mutant T42S, 25°C, pH not specified in the publication
0.015
fluoroacetyl-CoA
T42S mutant protein, pH not specified in the publication, 25°C
0.03
fluoroacetyl-CoA
wild-type, 25°C, pH not specified in the publication
0.03
fluoroacetyl-CoA
wild type protein, pH not specified in the publication, 25°C
0.206
fluoroacetyl-CoA
mutant E50A, 25°C, pH not specified in the publication
0.206
fluoroacetyl-CoA
E50A mutant protein, pH not specified in the publication, 25°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.044
fluoroacetyl-CoA
wild-type, 25°C, pH not specified in the publication
0.044
fluoroacetyl-CoA
wild type protein, pH not specified in the publication, 25°C
0.111
fluoroacetyl-CoA
mutant E50A, 25°C, pH not specified in the publication
0.111
fluoroacetyl-CoA
E50A mutant protein, pH not specified in the publication, 25°C
0.409
fluoroacetyl-CoA
mutant T42S, 25°C, pH not specified in the publication
0.409
fluoroacetyl-CoA
T42S mutant protein, pH not specified in the publication, 25°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.54
fluoroacetyl-CoA
mutant E50A, 25°C, pH not specified in the publication
0.54
fluoroacetyl-CoA
E50A mutant protein, pH not specified in the publication, 25°C
1.47
fluoroacetyl-CoA
wild-type, 25°C, pH not specified in the publication
1.47
fluoroacetyl-CoA
wild type protein, pH not specified in the publication, 25°C
27.3
fluoroacetyl-CoA
mutant T42S, 25°C, pH not specified in the publication
27.3
fluoroacetyl-CoA
T42S mutant protein, pH not specified in the publication, 25°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.8
acetyl-aza(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
4.7
acetyl-aza(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
8.4
acetyl-aza(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
33
acetyl-aza(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
2.6
acetyl-carba(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
3.6
acetyl-carba(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
11
acetyl-carba(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
51
acetyl-carba(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.3
acetyl-oxa(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.51
acetyl-oxa(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
1.5
acetyl-oxa(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
2.5
acetyl-oxa(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
1.2
fluoroacetyl-aza(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
4
fluoroacetyl-aza(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
4.3
fluoroacetyl-aza(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
7.5
fluoroacetyl-aza(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.46
fluoroacetyl-carba(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.9
fluoroacetyl-carba(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
2.1
fluoroacetyl-carba(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
4.5
fluoroacetyl-carba(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.13
fluoroacetyl-oxa(dethia)-CoA
wild type enzyme, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.26
fluoroacetyl-oxa(dethia)-CoA
mutant enzyme F36A, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.32
fluoroacetyl-oxa(dethia)-CoA
wild type enzyme, with fluoroacetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
0.33
fluoroacetyl-oxa(dethia)-CoA
mutant enzyme F36A, with acetyl-CoA as substrate, at pH 7.6, temperature not specified in the publication
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.0295
-
crude mitochondrial extract, pH not specified in the publication, temperature not specified in the publication
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
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presence of enzyme protects its host from fluoroacetate toxicity in vivo
Show AA Sequence and Transmembrane Helices (238 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, FlK employs a catalytic triad composed of Thr42, His76, and a water molecule
wild-type in complex with fluoroacetate, mutant F36A both alone and as product complex, to 2.3 A, 2.3 A, and 2.0 A resolution, respectively
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E50A
F36A
H76A
H76D
T42A
T42S
E50A
disturbed protein folding. 37% of wild-type catalytic efficiency, substrate inhibition above 0.019 mM
F36A
the mutation results in a change in KM by affecting substrate binding
H76A
the kinetic isotope effec observed with [2H2]fluoroacetyl-CoA is abolished in the H76A mutant, showing that H76 is directly involved in Calpha deprotonation
H76D
putative member of the catalytic triad, completely insoluble
T42S
disturbed protein folding. 19fold increase in catalytic efficiency. Substrate inhibition above 0.012 mM
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
immobilized metal ion affinity chromatography (Co2+), His tag removed, gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
His-tagged protein expressed in Escherichia coli Rosetta (DE3)pLysS
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Weeks, A.M.; Coyle, S.M.; Jinek, M.; Doudna, J.A.; Chang, M.C.
Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity
Biochemistry
49
9269-9279
2010
Streptomyces cattleya
brenda
Dias, M.V.; Huang, F.; Chirgadze, D.Y.; Tosin, M.; Spiteller, D.; Dry, E.F.; Leadlay, P.F.; Spencer, J.B.; Blundell, T.L.
Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK
J. Biol. Chem.
285
22495-22504
2010
Streptomyces cattleya (Q1EMV2), Streptomyces cattleya
brenda
Meyer, J.; Marion, J.; Vleggaar, R.; Louw, A.I.
Fluoroacetyl-coenzyme A hydrolase-like activity in Dichapetalum cymosum
J. Plant Physiol.
139
369-372
1992
Dichapetalum cymosum
-
brenda
Weeks, A.M.; Keddie, N.S.; Wadoux, R.D.; OHagan, D.; Chang, M.C.
Molecular recognition of fluorine impacts substrate selectivity in the fluoroacetyl-CoA thioesterase FlK
Biochemistry
53
2053-2063
2014
Streptomyces cattleya (Q1EMV2), Streptomyces cattleya
brenda
Weeks, A.M.; Chang, M.C.
Catalytic control of enzymatic fluorine specificity
Proc. Natl. Acad. Sci. USA
109
19667-19672
2012
Streptomyces cattleya (Q1EMV2), Streptomyces cattleya
brenda
Weeks, A.M.; Wang, N.; Pelton, J.G.; Chang, M.C.Y.
Entropy drives selective fluorine recognition in the fluoroacetyl-CoA thioesterase from Streptomyces cattleya
Proc. Natl. Acad. Sci. USA
115
E2193-E2201
2018
Streptomyces cattleya (Q1EMV2)
brenda
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Release 2023.1 (January 2023)
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