Information on EC 5.3.2.6 - 2-hydroxymuconate tautomerase

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

EC NUMBER
COMMENTARY hide
5.3.2.6
-
RECOMMENDED NAME
GeneOntology No.
2-hydroxymuconate tautomerase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate = (3E)-2-oxohex-3-enedioate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,3-dihydroxybenzoate degradation
-
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3-chlorocatechol degradation III (meta pathway)
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-
4-amino-3-hydroxybenzoate degradation
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4-chloronitrobenzene degradation
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Benzoate degradation
-
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catechol degradation to 2-oxopent-4-enoate II
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Dioxin degradation
-
-
Metabolic pathways
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Microbial metabolism in diverse environments
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orthanilate degradation
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protocatechuate degradation III (para-cleavage pathway)
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Xylene degradation
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phenol degradation
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SYSTEMATIC NAME
IUBMB Comments
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate keto-enol isomerase
Involved in the meta-cleavage pathway for the degradation of phenols, modified phenols and catechols. The enol form (2Z,4E)-2-hydroxyhexa-2,4-dienedioate is produced as part of this pathway and is converted to the keto form (3E)-2-oxohex-3-enedioate by the enzyme [6]. Another keto form, (4E)-2-oxohex-4-enedioate (4-oxalocrotonate), was originally thought to be produced by the enzyme [1,2] but later shown to be produced non-enzymically [5].
CAS REGISTRY NUMBER
COMMENTARY hide
85876-28-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
two isozymes
UniProt
Manually annotated by BRENDA team
two isozymes
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
formerly Bacillus macerans, gene praC
UniProt
Manually annotated by BRENDA team
formerly Bacillus macerans, gene praC
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(1Z)-1-(nitromethylidene)-3a,4,5,6,7,7a-hexahydro-1H-indene + acetaldehyde
[1-(nitromethyl)-1H-inden-1-yl]acetaldehyde
show the reaction diagram
-
-
-
-
?
(1Z)-4-methyl-1-nitropent-1-ene + acetaldehyde
5-methyl-3-(nitromethyl)hexanal
show the reaction diagram
-
-
-
-
?
(2E)-2-hydroxy-3-phenylprop-2-enoate
2-oxo-3-phenylpropanoate
show the reaction diagram
-
-
-
-
?
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
(2E)-5-oxohex-2-enedioate
show the reaction diagram
-
-
-
?
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
(2E)-5-oxohex-2-enedioic acid
show the reaction diagram
-
-
-
-
?
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
(3E)-2-oxohex-3-enedioate
show the reaction diagram
(3E)-6-oxohept-3-enedioate
2-hydroxy-2,4-heptadiene-1,7-dioate
show the reaction diagram
-
-
-
-
?
1-chloro-4-[(Z)-2-nitroethenyl]benzene + acetaldehyde
3-(4-chlorophenyl)-4-nitrobutanal
show the reaction diagram
-
-
-
-
?
1-fluoro-4-[(Z)-2-nitroethenyl]benzene + acetaldehyde
3-(4-fluorophenyl)-4-nitrobutanal
show the reaction diagram
-
-
-
-
?
2-(cyclopentyloxy)-1-methoxy-4-[(Z)-2-nitroethenyl]benzene + acetaldehyde
3-[3-(cyclopentyloxy)-4-methoxyphenyl]-4-nitrobutanal
show the reaction diagram
-
-
-
-
?
2-chloro-beta-nitrostyrene + acetaldehyde
3-(2-chlorophenyl)-4-nitrobutanal
show the reaction diagram
-
-
51% yield
-
?
2-hydroxy-2,4-heptadiene-1,7-dioate
(3E)-2-oxohept-3-enedioate
show the reaction diagram
-
-
-
-
?
2-hydroxy-2,4-hexadienedioate
2-oxo-3,4-hexenedioate
show the reaction diagram
-
4-OT with 2-hydroxy-2,4-hexadienedioate in D2O results in a racemic mixture of 2-oxo-[3-2H]-4-hexenedioate, suggesting that 4-OT may not catalyze a 1,3-keto-enol tautomerization reaction using this dienol
-
-
?
2-hydroxy-2,4-hexadienedioate
2-oxo-3-hexenedioate
show the reaction diagram
2-hydroxy-2,4-pentadienoate
(2E)-5-oxohex-2-enedioate
show the reaction diagram
-
1,3- and 1,5-keto-enol tautomerization reactions are observed for 2-hydroxy-2,4-hexadienedioate. YwhB converts it to the 3S isomer of 2-oxo-4-hexenedioate in a highly stereoselective manner
main product
-
?
2-hydroxy-2,4-pentadienoate
(3E)-2-oxopent-3-enoate
show the reaction diagram
-
-
-
-
?
2-hydroxy-2,4-pentadienoate
2-oxo-3,4-pentenoate
show the reaction diagram
-
enzyme-catalyzed ketonization of 2-hydroxy-2,4-pentadienoate to (3R)-2-oxo-[3-D]-4-pentenoate is only stereoselective
-
-
?
2-hydroxy-2,4-pentadienoate
2-oxo-4-pentenoate
show the reaction diagram
2-hydroxy-2,4-pentadienoate
2-oxopent-3-enoic acid
show the reaction diagram
-
-
-
-
?
2-hydroxy-4-trans-hexenedioate
?
show the reaction diagram
-
-
-
-
?
2-hydroxymuconate
2-oxo-3-(E)-hexenedioate
show the reaction diagram
2-hydroxymuconate
2-oxo-3-hexenedioate
show the reaction diagram
2-hydroxymuconate
4-oxalocrotonate
show the reaction diagram
2-methoxy-5-[(Z)-2-nitroethenyl]phenol + acetaldehyde
3-(3-hydroxy-4-methoxyphenyl)-4-nitrobutanal
show the reaction diagram
-
-
-
-
?
2-oxo-3-hexenedioate
2-oxo-3-trans-hexenedioate
show the reaction diagram
-
-
-
-
?
2-oxo-4(E)-hexenedioate
2-oxo-3(E)-hexenedioate
show the reaction diagram
-
1,3-allylic isomerization
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-
?
2-oxo-4-hexenedioate
2-oxo-3-hexenedioate
show the reaction diagram
2-oxopent-4-enoate
2-hydroxy-2,4-pentadienoate
show the reaction diagram
-
-
-
-
?
4-chloro-beta-nitrostyrene + acetaldehyde
3-(4-chlorophenyl)-4-nitrobutanal
show the reaction diagram
-
-
38% yield
-
?
4-fluoro-beta-nitrostyrene + acetaldehyde
3-(4-fluorophenyl)-4-nitrobutanal
show the reaction diagram
-
-
31% yield
-
?
4-vinyl-2,3-dihydropyrrole-2-carboxylic acid
4-ethylidene-3,4-dihydropyrrole-2-carboxylic acid
show the reaction diagram
-
-
-
-
r
5-(methyl)-2-hydroxymuconate
5-(methyl)-2-oxo-3-hexenedioate
show the reaction diagram
-
-
-
?
acetaldehyde + [(Z)-2-nitroethenyl]benzene
(3S)-4-nitro-3-phenylbutanal
show the reaction diagram
-
-
-
-
?
beta-nitrostyrene + acetaldehyde
4-nitro-3-phenyl-butanal
show the reaction diagram
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60% yield
-
?
butanal + [(Z)-2-nitroethenyl]benzene
(2R,3S)-2-ethyl-4-nitro-3-phenylbutanal
show the reaction diagram
-
-
-
-
?
heptanal + [(Z)-2-nitroethenyl]benzene
(2R)-2-[(1S)-2-nitro-1-phenylethyl]heptanal
show the reaction diagram
-
-
-
-
?
hexanal + [(Z)-2-nitroethenyl]benzene
(2R)-2-[(1S)-2-nitro-1-phenylethyl]hexanal
show the reaction diagram
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-
-
-
?
octanal + [(Z)-2-nitroethenyl]benzene
(2R)-2-[(1S)-2-nitro-1-phenylethyl]octanal
show the reaction diagram
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-
-
-
?
pentanal + [(Z)-2-nitroethenyl]benzene
(2R)-2-[(1S)-2-nitro-1-phenylethyl]pentanal
show the reaction diagram
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-
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?
phenylenolpyruvate
phenylpyruvate
show the reaction diagram
propanal + [(Z)-2-nitroethenyl]benzene
(2R,3S)-2-methyl-4-nitro-3-phenylbutanal
show the reaction diagram
-
-
-
-
?
[(Z)-2-nitroethenyl]benzene + acetaldehyde
4-nitro-3-phenylbutanal
show the reaction diagram
-
-
-
-
?
additional information
?
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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
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
(3E)-2-oxohex-3-enedioate
show the reaction diagram
2-hydroxy-2,4-hexadienedioate
2-oxo-3-hexenedioate
show the reaction diagram
2-hydroxy-2,4-pentadienoate
2-oxopent-3-enoic acid
show the reaction diagram
-
-
-
-
?
2-hydroxymuconate
2-oxo-3-hexenedioate
show the reaction diagram
2-hydroxymuconate
4-oxalocrotonate
show the reaction diagram
2-oxo-4(E)-hexenedioate
2-oxo-3(E)-hexenedioate
show the reaction diagram
-
1,3-allylic isomerization
-
-
?
2-oxo-4-hexenedioate
2-oxo-3-hexenedioate
show the reaction diagram
phenylenolpyruvate
phenylpyruvate
show the reaction diagram
-
-
-
-
?
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2E)-2-fluoro-2,4-pentadienoate
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-
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(2E)-fluoromuconate
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a dicarboxylate competitive inhibitor, shows substoichiometric binding to 3 +/- 1 sites per hexamer
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(2Z)-2-fluoro-2,4-pentadienoate
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-
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2-Oxo-3-pentynoate
3-Bromopyruvate
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a active-site-directed irreversible inhibitor, modification of three active sites per hexamer abolishes essentially all activity of the hexamer, pH dependence of inactivation
additional information
-
although 4-OT exhibits no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels shows half-site stoichiometry
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.334
(2E)-2-hydroxy-3-phenylprop-2-enoate
-
wild type enzyme, in 10 mM potassium phosphate buffer, pH 7.3, at 22°C
0.11 - 0.17
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
0.017 - 1.6
2-hydroxy-2,4-hexadienedioate
0.09 - 1.11
2-hydroxy-2,4-pentadienoate
0.017 - 1.05
2-hydroxymuconate
0.189
2-oxo-3-hexenedioate
-
pH 7.3, 30°C
0.09 - 0.103
2-oxo-4(E)-hexenedioate
0.105 - 0.211
5-(methyl)-2-hydroxymuconate
0.063 - 0.23
phenylenolpyruvate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
16
(2E)-2-hydroxy-3-phenylprop-2-enoate
Pseudomonas putida
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wild type enzyme, in 10 mM potassium phosphate buffer, pH 7.3, at 22°C
0.05 - 0.6
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
0.4 - 3500
2-hydroxy-2,4-hexadienedioate
0.1 - 2.5
2-hydroxy-2,4-pentadienoate
0.2 - 139000
2-hydroxymuconate
288000
2-oxo-3-hexenedioate
Pseudomonas putida
-
pH 7.3, 30°C
2890 - 2940
2-oxo-4(E)-hexenedioate
3.4 - 107
5-(methyl)-2-hydroxymuconate
0.2 - 44
phenylenolpyruvate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
48
(2E)-2-hydroxy-3-phenylprop-2-enoate
Pseudomonas putida
-
wild type enzyme, in 10 mM potassium phosphate buffer, pH 7.3, at 22°C
193943
0.45 - 5.4
(2Z,4E)-2-hydroxyhexa-2,4-dienedioate
5068
19 - 51000
2-hydroxy-2,4-hexadienedioate
3048
0.36 - 13
2-hydroxy-2,4-pentadienoate
6375
0.45 - 957000
2-hydroxymuconate
4290
1520000
2-oxo-3-hexenedioate
Pseudomonas putida
-
pH 7.3, 30°C
8543
29000 - 32000
2-oxo-4(E)-hexenedioate
19684
16 - 1000
5-(methyl)-2-hydroxymuconate
194881
3.2 - 290
phenylenolpyruvate
3296
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.045
(2E)-fluoromuconate
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pH and temperature not specified in the publication
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.75
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7732
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits
7963
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits
8096
3* 7730, alpha-subunit, + 3 * 7963, beta-subunit, mass spectrometry, 3 * 7732, alpha-subunit + 3 * 8096, beta-subunit, sequence calculation, functional heterohexamer OT, hh4-OT, composed of three alphabeta dimers, the fully functional enzyme requires both subunits
10000
-
x * 10000, SDS-PAGE
14000
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x * 14000, 5-(carboxymethyl)-2-hydroxymuconate isomerase, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterohexamer
hexamer
homohexamer
-
-
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals are grown from 20% dioxane and 50% 2-methyl-2,4-pentanediol, pH 6.0, vapor diffusion method
purified recombinant hh4-OT, by sitting drop vapour diffusion method, 0.001 ml of 20 mg/mL protein in 10 mM Na/KPO4 buffer, pH 7.3, is mixed in a 1:1 ratio with precipitant against a 0.1 ml reservoir solution, and by hanging drop vapor diffusion method with a 0.5 ml reservoir and 0.004 ml hanging drops that contain protein and precipitant, 0.25 M (NH4)2SO4 and 4% PEG 4000, in a 1:1 ratio, 3-5 days, X-ray diffraction structure determination and analysis at 2.41 A resolution
crystals are grown from 28% polyethylene glycol 400, 200 mM CaCl2, 0.1 M HEPES buffer (pH 7.5), vapor diffusion method
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hanging drop vapor diffusion method, using 200 mM tri-ammonium citrate and 10% (w/v)polyethylene glycol (PEG) 3350
sitting drop vapor diffusion method, using 100 mM magnesium acetate/100 mM sodium acetate, 5%-21% (w/v) PEG 8000, pH 4.5, and 100 mM calcium acetate/100 mM sodium acetate, 1%-13% (w/v) PEG 4000, pH 4.5
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4-OT from 80% saturated ammonium sulfate solution containing 100 mM HEPES, pH 7.5, and 1% PEG 200, 20°C, X-ray diffraction structure determination and analysis at 1.9-2.8 A resolution, heavy etal derivatization by soaking the crystals for 4 h in crystallization solution containing 2 mM K2PtCl4
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
higher thermostability of the hh4-OT for stability in the thermophilic environment of the organism, overview
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sepharose column chromatography and SP-Sepharose column chromatography
native enzyme from Escherichia coli strain C by ammonium sulfate fractionation, gel filtration, anion exchange chromatography, ultrafiltration, ammoniums ulfate fractionation, and hydrophobic interaction chromatography, followed by gel filtration
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phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography
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recombinant enzyme from Escherichia coli by ultracentrifugation, two different steps of anion exchange chromatography, hydroxylapatite and hydrophobic interaction chromatography, and gel filtration with alternating steps of ultrafiltration
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recombinant hh4-OT wild-type and mutants from Escherichia coli by heat treatment, anion exchange chromatography, and gel-filtration
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21-Gold(DE3) to over 95% purity
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recombinant wild-type and mutant YwhBs from Escherichia coli strain BL21(DE3)pLysS
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recombinant wild-type and mutants from Escherichia coli strain BL21(DE3)pLysS to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloning of alpha- and beta-subunits of hh4-OT from genomic DNA, recombinant overexpression of wild-type hh4-OT, and of mutants in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-Gold(DE3) cells
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expression of wild-type and mutant YwhBs in Escherichia coli strain BL21(DE3)pLysS
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gene praC, DNA and amino acid sequence determination and analysis, expression in Escherichia coli BL21(DE3)
overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21-Gold(DE3)
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overexpression of wild-type and mutants in Escherichia coli strain BL21(DE3)pLysS, subcloning in Escherichia coli strain JM109
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recombinant expression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P1A
-
site-directed mutagenesis, inactive mutant
R11A
-
site-directed mutagenesis, inactive mutant
alphaR12A
site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT
alphaR40A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT
betaP1A
site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT
betaR11A
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type hh4-OT
betaR39A
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT
alphaR12A
-
site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT
-
alphaR40A
-
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT
-
betaP1A
-
site-directed mutagenesis, the mutant shows highly reduced catalytic efficiency compared to the wild-type hh4-OT
-
betaR11A
-
site-directed mutagenesis, the mutant shows increased catalytic efficiency compared to the wild-type hh4-OT
-
betaR39A
-
site-directed mutagenesis, the mutant shows reduced catalytic efficiency compared to the wild-type hh4-OT
-
alphaI52E
-
site-directed mutagenesis, active site mutant,the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 36fold increase in kcat/Km, largely due to a 110fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity. The negatively charged group may hinder the formation of the enolate intermediate and may contribute to a decrease in kcat
alphaL8R
-
site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 50fold increase in kcat/Km, primarily from an 8.8fold increase in kcat, and diminished 4-oxalocrotonate tautomerase activity with a 5fold decrease in kcat/Km. The increased CaaD activity of L8R-4-OT does not substantially diminish the original 4-OT activity
alphaL8R/I52E
-
site-directed mutagenesis, active site mutant, the mutant shows improved trans-3-chloroacrylic acid dehalogenase activity with a 32fold increase in kcat/Km, largely due to a 23fold decrease in Km, and diminished 4-oxalocrotonate tautomerase activity with a 1700fold decrease in kcat/Km
P1A
-
the mutation results in 430fold decreases in kcat/Km compared to the wild type enzyme
R11A/R39A
-
site-directed mutagenesis, inactive mutant
Show AA Sequence (349 entries)
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