Information on EC 5.99.1.4 - 2-hydroxychromene-2-carboxylate isomerase

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The expected taxonomic range for this enzyme is: Proteobacteria

EC NUMBER
COMMENTARY hide
5.99.1.4
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RECOMMENDED NAME
GeneOntology No.
2-hydroxychromene-2-carboxylate isomerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-hydroxy-2H-chromene-2-carboxylate = (3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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Microbial metabolism in diverse environments
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Naphthalene degradation
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naphthalene degradation (aerobic)
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SYSTEMATIC NAME
IUBMB Comments
2-hydroxy-2H-chromene-2-carboxylate-(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate isomerase
This enzyme is involved in naphthalene degradation.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
BN6, DSM 6383
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Manually annotated by BRENDA team
A3, DSM 676
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Manually annotated by BRENDA team
strain TA-2
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Manually annotated by BRENDA team
strain TA-2
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Manually annotated by BRENDA team
BN6
SwissProt
Manually annotated by BRENDA team
BN6
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
2-hydroxybenzo[g]chromene-2-carboxylate
?
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
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glutathione
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the dimeric protein binds one molecule of GSH very tightly and a second molecule of GSH with much lower affinity. The enzyme is unstable in the absence of GSH. The turnover number in the forward direction greatly exceeds off rates for GSH, suggesting that GSH acts as a tightly bound cofactor in the reaction. Km: 0.017 mM
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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i.e. (E)-2'-hydroxybenzylidenepyruvate
iodoacetic acid
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monoiodoacetate
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additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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dithiothreitol
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glutathione
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.138
(E)-2'-hydroxybenzylidenepyruvate
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pH 7.0, 25C
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0.053 - 0.27
2-hydroxy-2H-chromene-2-carboxylate
0.084 - 0.23
2-hydroxychromene-2-carboxylate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
19
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
Pseudomonas putida
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pH 7.0, 25C
47
2-hydroxy-2H-chromene-2-carboxylate
Pseudomonas putida
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pH 7.0, 25C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.136
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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pH 7.0, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.14
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for 2-hydroxychromene-2-carboxylate as substrate
0.27
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for 2-hydroxychromene-2-carboxylate as substrate
55.4
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last purification step
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
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in glycine-NaOH-buffer, highest enzyme activity is found after preincubation of the enzyme with glutathione at alkaline pH-values
9.5
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in glycine-NaOH-buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 9.5
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at pH 9.0 50% and at pH 8.0 only 5% of the maximal activity is found, respectively
8 - 9.3
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effect of glutathione is optimal between pH 8 and pH 9.3. In contrast, no activation is observed when the enzyme is incubated with glutathione at pH 5.0. Activities higher than 70% of this optimal activity are found at pH-values in the range 8.0-9.5 in glycine-NaOH- or Tris/HC1-buffer. At pH 7 about 50% and at pH 5.5 10% of the optimal activity is observed
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8 - 9.5
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pH 6.8: about 35% of maximal activity, pH 9.5: about 40% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5
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isoelectric focusing
5.4
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calculated from sequence
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 23031, calculated from sequence
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the enzyme at 1.7 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
1.5 - 10
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4C, 40 h, stable in the range of pH 1.5 to 10.0
698643
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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10 min, stable at temperature up to
50
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10 min, in presence of glutathione at 2.5 mM, the enzyme is stable up to
55
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10 min, 13% of the original activity remains. In presence of glutathione at 2.5 mM, 68% of the original activity remains after 10 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable in absence of GSH
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, purifed enzyme, Tris HCl, pH 7.5, 100 mM NaCl, 1 month 52% loss of activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at room temperature by use of a fast-protein liquid chromatography system
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partially purified by anion-exchange chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a gene cluster is identified on the plasmid pBN6 which codes for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16915 bp is sequenced which contains 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 29-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway are identified on the DNA fragment and the encoded proteins are heterologously expressed in Escherichia coli
expression in Escherichia coli
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