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Information on EC 5.99.1.4 - 2-hydroxychromene-2-carboxylate isomerase
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5.99.1.4 2-hydroxychromene-2-carboxylate isomeraseIUBMB Comments
This enzyme is involved in naphthalene degradation.
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The enzyme appears in viruses and cellular organisms
Reaction Schemes
Synonyms
2-hydroxychromene-2-carboxylate isomerase, 2hc2ca isomerase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
2-hydroxychromene-2-carboxylate isomerase
2HC2CA isomerase
2HCCAI
nsaD
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2-hydroxy-2H-chromene-2-carboxylate = (3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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PATHWAY SOURCE
PATHWAYS
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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.
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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
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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?
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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?
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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
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r
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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
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?
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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the product trans-o-hydroxybenzylidenepyruvate is analyzed by 1H NMR spectrum
i.e. (E)-2'-hydroxybenzylidenepyruvate, i.e. trans-o-hydroxybenzylidenepyruvate
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?
2-hydroxy-2H-chromene-2-carboxylate
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
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the product trans-o-hydroxybenzylidenepyruvate is analyzed by 1H NMR spectrum
i.e. (E)-2'-hydroxybenzylidenepyruvate, i.e. trans-o-hydroxybenzylidenepyruvate
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?
2-hydroxybenzo[g]chromene-2-carboxylate
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more unstable than 2-hydroxychromene-2-carboxylate
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2-hydroxybenzo[g]chromene-2-carboxylate
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more unstable than 2-hydroxychromene-2-carboxylate
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?
additional information
?
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enzyme degrades naphthalenesulfonates
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?
additional information
?
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2,5-dihydroxychromene-2-carboxylate is no substrate
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?
additional information
?
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enzyme degrades naphthalenesulfonates
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?
additional information
?
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2,5-dihydroxychromene-2-carboxylate is no 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
additional information
?
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enzyme degrades naphthalenesulfonates
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?
additional information
?
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enzyme degrades naphthalenesulfonates
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?
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COFACTOR
ORGANISM
UNIPROT
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
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
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no increase of enzyme activity is found after (pre)incubation of the enzyme with ZnSO4, COCl2, FeCl2 or MnCl2 (each 2 mM)
additional information
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no increase of enzyme activity is found after (pre)incubation of the enzyme with ZnSO4, COCl2, FeCl2 or MnCl2 (each 2 mM)
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(3E)-4-(2-hydroxyphenyl)-2-oxobut-3-enoate
i.e. (E)-2'-hydroxybenzylidenepyruvate
additional information
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no significant inhibition is observed after one day incubation with 50 mM EDTA
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additional information
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no significant inhibition is observed after one day incubation with 50 mM EDTA
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
glutathione
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no enzyme activity is found when the enzyme preparations have been preincubated without or with only 10 mM glutathione
glutathione
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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
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.005
0.2
0.2
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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
0.2
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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
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
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
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
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
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Show AA Sequence and Transmembrane Helices (10914 entries)
Longer loading times are possible. Please use the AA Sequence and Transmembrane Helices Search for a specific query.
Longer loading times are possible. Please use the AA Sequence and Transmembrane Helices Search for a specific query.
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
24700
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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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
4C, purifed enzyme, Tris HCl, pH 7.5, 100 mM NaCl, 1 month 52% loss of activity
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4C, purifed enzyme, Tris HCl, pH 7.5, 100 mM NaCl, 1 month 52% loss of activity
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
at room temperature by use of a fast-protein liquid chromatography system
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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
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Eaton, R.W.; Chapman, P.J.
Bacterial metabolism of naphthalene: Construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions
J. Bacteriol.
174
7542-7554
1992
Pseudomonas putida (Q51948)
brenda
Thompson, L.C.; Ladner, J.E.; Codreanu, S.G.; Harp, J.; Gilliland, G.L.; Armstrong, R.N.
2-Hydroxychromene-2-carboxylic acid isomerase: a kappa class glutathione transferase from Pseudomonas putida
Biochemistry
46
6710-6722
2007
Pseudomonas putida (Q51948)
brenda
R. W., Eaton
Organization and evolution of naphthalene catabolic pathways: sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NAH7 plasmid
J. Bacteriol.
176
7757-7762
1994
Pseudomonas putida (Q51948)
brenda
Ohmoto, T.; Kinoshita, T.; Moriyoshi, K.; Sakai, K.; Hamada, N.; Ohe, T.
Purification and some properties of 2-hydroxychromene-2-carboxylate isomerase from naphthalenesulfonate-assimilating Pseudomonas sp. TA-2
J. Biochem.
124
591-597
1998
Pseudomonas sp., Pseudomonas sp. TA-2
brenda
Keck, A.; Conradt, D.; Mahler, A.; Stolz, A.; Mattes, R.; Klein, J.
Identification and functional analysis of the genes for naphthalenesulfonate catabolism by Sphingomonas xenophaga BN6
Microbiology
152
1929-1940
2006
Sphingobium xenophagum (Q9X9Q7), Sphingobium xenophagum, Sphingobium xenophagum BN6 (Q9X9Q7)
brenda
Kuhm, A.E.; Knackmuss, H.-J.; Stolz, A.
2-Hydroxychromene-2-carboxylate isomerase from bacteria that degrade naphthalenesulfonates
Biodegradation
4
155-162
1993
Brevundimonas vesicularis, Comamonas testosteroni
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brenda
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