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

Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Mark a special word or phrase in this record:
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Proteobacteria

EC NUMBER
COMMENTARY
5.99.1.4
-
RECOMMENDED NAME
GeneOntology No.
2-hydroxychromene-2-carboxylate isomerase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2-hydroxy-2H-chromene-2-carboxylate = (3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Metabolic pathways
-
Microbial metabolism in diverse environments
-
Naphthalene degradation
-
naphthalene degradation (aerobic)
-
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.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2-hydroxychromene-2-carboxylate isomerase
-
-
2-hydroxychromene-2-carboxylate isomerase
Q51948
-
2-hydroxychromene-2-carboxylic acid isomerase
Q51948
-
2HC2CA isomerase
-
-
2HC2CA isomerase
Pseudomonas sp. TA-2
-
-
-
HCCA isomerase
Q51948
-
nsaD
Sphingobium xenophagum BN6
Q9X9Q7
-
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
BN6, DSM 6383
-
-
Manually annotated by BRENDA team
A3, DSM 676
-
-
Manually annotated by BRENDA team
strain TA-2
-
-
Manually annotated by BRENDA team
Pseudomonas sp. TA-2
strain TA-2
-
-
Manually annotated by BRENDA team
Sphingobium xenophagum BN6
BN6
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
Q51948
extracts of Escherichia coli JM109 carrying pRE718 catalyze the conversion of trans-o-hydroxybenzylidenepyruvate or 2-hydroxychromene-2-carboxylate to an equilibrium mixture that contained 55% 2-hydroxychromene-2-carboxylate and 45% trans-o-hydroxybenzylidenepyruvate at pH 7. At pH 10 the reaction occus entirely in on direction, the conversion of 2-hydroxychromene-2-carboxylate to trans-o-hydroxybenzylidenepyruvate. The product is identified by nuclear magnetic resonance spectroscopy. This isomerization occurs spontaneously, although at a slower rate than the enzyme-catalyzed reaction
i.e. (E)-2'-hydroxybenzylidenepyruvate, i.e. trans-o-hydroxybenzylidenepyruvate
-
r
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
-
glutathione (GSH)-dependent interconversion. The isomerization reaction involves a short-lived covalent adduct between the sulfur of GSH and C7 of the substrate
i.e. (E)-2'-hydroxybenzylidenepyruvate, i.e. trans-o-hydroxybenzylidenepyruvate
-
?
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
show the reaction diagram
Pseudomonas sp., Pseudomonas sp. TA-2
-
the product trans-o-hydroxybenzylidenepyruvate is analyzed by 1H NMR spectrum
i.e. (E)-2'-hydroxybenzylidenepyruvate, i.e. trans-o-hydroxybenzylidenepyruvate
-
?
2-hydroxybenzo[g]chromene-2-carboxylate
?
show the reaction diagram
-
more unstable than 2-hydroxychromene-2-carboxylate
-
-
?
additional information
?
-
-
enzyme degrades naphthalenesulfonates
-
-
-
additional information
?
-
-
2,5-dihydroxychromene-2-carboxylate is no substrate
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
enzyme degrades naphthalenesulfonates
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
glutathione
-
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 ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
no increase of enzyme activity is found after (pre)incubation of the enzyme with ZnSO4, COCl2, FeCl2 or MnCl2 (each 2 mM)
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
-
i.e. (E)-2'-hydroxybenzylidenepyruvate
iodoacetic acid
-
-
monoiodoacetate
-
-
additional information
-
no significant inhibition is observed after one day incubation with 50 mM EDTA
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2-mercaptoethanol
-
-
glutathione
-
activates above 0.15 mM
glutathione
-
no enzyme activity is found when the enzyme preparations have been preincubated without or with only 10 mM glutathione
glutathione
-
highest enzyme activity is found after preincubation of the enzyme with glutathione at alkaline pH-values. To determine enzyme activities during the purification procedure, it is necessary to reactivate the enzyme with glutathione
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.138
-
(E)-2'-hydroxybenzylidenepyruvate
-
pH 7.0, 25C
-
0.053
-
2-hydroxy-2H-chromene-2-carboxylate
-
pH 7.4, 30C
0.27
-
2-hydroxy-2H-chromene-2-carboxylate
-
-
0.084
-
2-hydroxychromene-2-carboxylate
-
pH 7.0, 25C
-
0.23
-
2-hydroxychromene-2-carboxylate
-
-
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
19
-
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
-
pH 7.0, 25C
47
-
2-hydroxy-2H-chromene-2-carboxylate
-
pH 7.0, 25C
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.136
-
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
-
pH 7.0, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.005
-
-
spectrophotometrically
0.14
-
-
for 2-hydroxychromene-2-carboxylate as substrate
0.2
-
-
enzyme activity increases when the cell extract is incubated for 15 min with glutathione (5 mM) prior to the assay. The addition of glutathione (0.2 mM) directly to the assay does not increase enzyme activity
0.27
-
-
for 2-hydroxychromene-2-carboxylate as substrate
55.4
-
-
last purification step
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
9
-
-
in glycine-NaOH-buffer, highest enzyme activity is found after preincubation of the enzyme with glutathione at alkaline pH-values
9.5
-
-
in glycine-NaOH-buffer
10
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
9.3
-
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
8
9.5
-
at pH 9.0 50% and at pH 8.0 only 5% of the maximal activity is found, respectively
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.8
9.5
-
pH 6.8: about 35% of maximal activity, pH 9.5: about 40% of maximal activity
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5
-
-
isoelectric focusing
5.4
-
-
calculated from sequence
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
27000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 23031, calculated from sequence
monomer
-
1 * 25000, SDS-PAGE
monomer
Pseudomonas sp. TA-2
-
1 * 25000, SDS-PAGE
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystal structure of the enzyme at 1.7 A resolution
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
1.5
10
-
4C, 40 h, stable in the range of pH 1.5 to 10.0
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
45
-
-
10 min, stable at temperature up to
50
-
-
10 min, in presence of glutathione at 2.5 mM, the enzyme is stable up to
55
-
-
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 ACCESSION NO.
LITERATURE
unstable in absence of GSH
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4C, purifed enzyme, Tris HCl, pH 7.5, 100 mM NaCl, 1 month 52% loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
partially purified by anion-exchange chromatography
-
at room temperature by use of a fast-protein liquid chromatography system
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
-
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
Q9X9Q7, -