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Information on EC 4.1.3.42 - (4S)-4-hydroxy-2-oxoglutarate aldolase and Organism(s) Escherichia coli and UniProt Accession P0A955
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4.1.3.42 (4S)-4-hydroxy-2-oxoglutarate aldolaseIUBMB Comments
The enzyme from the bacterium Escherichia coli, which is specific for the (S)-enantiomer, is trifunctional, and also catalyses the reaction of EC 4.1.2.14, 2-dehydro-3-deoxy-phosphogluconate aldolase, and the beta-decarboxylation of oxaloacetate. cf. EC 4.1.3.16, 4-hydroxy-2-oxoglutarate aldolase.
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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
2-keto-4-hydroxybutyrate aldolase, 2-keto-4-hydroxyglutarate aldolase, 2-keto-4-hydroxyglutaric aldolase, 2-oxo-4-hydroxyglutarate aldolase, 2-oxo-4-hydroxyglutaric aldolase, 4-hydroxy-2-ketoglutarate aldolase, 4-hydroxy-2-ketoglutaric aldolase, 4-hydroxy-2-oxoglutarate glyoxylate-lyase, eda, HGA, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
(4S)-4-hydroxy-2-oxoglutarate glyoxylate-lyase (pyruvate-forming)
The enzyme from the bacterium Escherichia coli, which is specific for the (S)-enantiomer, is trifunctional, and also catalyses the reaction of EC 4.1.2.14, 2-dehydro-3-deoxy-phosphogluconate aldolase, and the beta-decarboxylation of oxaloacetate. cf. EC 4.1.3.16, 4-hydroxy-2-oxoglutarate aldolase.
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
(4R)-4-hydroxy-2-oxoglutarate
pyruvate + glyoxylate
poor substrate
-
-
r
(4S)-4-hydroxy-2-oxobutanoate
formaldehyde
at 8% of the rate with DL-4-hydroxy-2-oxoglutarate
-
-
r
D,L-4-hydroxy-2-oxoglutarate
pyruvate + glyoxylate + D-4-hydroxy-2-oxoglutarate
-
-
preferential cleavage of the L-isomer of racemic 4-hydroxy-2-oxoglutarate leaving the D-isomer in solution
-
r
Glyoxylate + pyruvate
4-Hydroxy-2-ketoglutarate
higher specificity for pyruvate than for glyoxylate
-
-
?
pyruvate + glyoxylate
(4S)-4-hydroxy-2-oxoglutarate
-
-
i.e. L-4-hydroxy-2-oxoglutarate. Condensation of pyruvate and glyoxylate carried out at a relatively high concentration leads to a quantitative conversion of pyruvate and glyoxylate to (4S)-4-hydroxy-2-oxoglutarate
-
r
(4S)-4-hydroxy-2-oxoglutarate
pyruvate + glyoxylate
-
L-4-hydroxy-2-oxoglutarate, pyruvate and glyoxylate all bind at the same active site of the enzyme
-
r
2-Keto-4-hydroxybutyrate
Pyruvate + formaldehyde
-
8% of 4-hydroxy-2-oxoglutarate cleavage rate
-
-
?
4-hydroxy-2-oxoglutarate
?
-
involved respiration recovery after RecA-dependent SOS response
-
-
?
4-hydroxy-2-oxoglutarate
?
possibly involved in regulation of tricarboxylic acid cycle
-
-
?
additional information
?
-
-
enzyme additionally shows beta-decarboxylase activity towards oxalacetate
-
-
?
additional information
?
-
enzyme additionally shows beta-decarboxylase activity towards oxalacetate
-
-
?
additional information
?
-
enzyme binds any one of its substrates 2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate in a competitive manner
-
-
?
additional information
?
-
-
residue Glu45 is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor
-
-
?
additional information
?
-
residue Glu45 is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor
-
-
?
additional information
?
-
-
ratio of stereoisomers cleaved approaches 1 during prolonged incubation, mechanism of stereospecificity
-
-
?
additional information
?
-
-
enzyme shows high specificity for both glyoxylate and pyruvate
-
-
?
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
4-hydroxy-2-oxoglutarate
?
-
involved respiration recovery after RecA-dependent SOS response
-
-
?
4-hydroxy-2-oxoglutarate
?
possibly involved in regulation of tricarboxylic acid cycle
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
divalent kations are not required. No effect on activity: Mg2+, Mn2+, Zn2+, Ca2+, Ba2+, Co2+, Cu2+, Ni2+, Fe2+, Hg2+, K+, or Fe3+
additional information
divalent kations are not required. No effect on activity: Mg2+, Mn2+, Zn2+, Ca2+, Ba2+, Co2+, Cu2+, Ni2+, Fe2+, Hg2+, K+, or Fe3+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Bromopyruvate
substrate analog. Treatment results in a time- and concentration-dependent loss of enzymatic activity. The substrates pyruvate and 2-keto-4-hydroxyglutarate provide more than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde. 1.1 mol of bromopyruvate is incorporated per enzyme subunit by esterification of residue Glu45
CN-
irreversible loss of activity in presence of glyoxylate, but not in presence of pyruvate
glyoxylate
substrate inhibition. Glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit
NaBH4
extensive loss of activity after incubation of enzyme with NaBH4 in presence of pyruvate or glyoxylate
4-(Oxyacetyl)phenoxyacetic acic
-
inactivates arginine residues, 90% inactivation at 10 mM
Bromopyruvate
85% inhibition at 1 mM, pyruvate or 2-keto-4-hydroxyglutarate protect
glyoxylate
-
bovine enzyme inhibited at lower concentrations than E. coli enzyme
additional information
-
not inhibitory: EDTA, 1,10-phenanthroline, S-hydroxyquinoline, or alpha,alpha'-dipyridyl
-
additional information
not inhibitory: EDTA, 1,10-phenanthroline, S-hydroxyquinoline, or alpha,alpha'-dipyridyl
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.45
(4S)-4-hydroxy-2-oxoglutarate
-
pH 8.4, temperature not specified in the publication
3.5
(4S)-4-hydroxy-2-oxoglutarate
-
enzyme reactivated after acidic denaturation, pH 8.4, temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
80fold increase in specific activity after overexpression in pKK223.3
additional information
80fold increase in specific activity after overexpression in pKK223.3
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8.6
additional information
additional information
-
2.2 to 2.4 times higher activity is measured in Tris-HCl buffer than in potassium phosphate buffer at the same pH values
additional information
2.2 to 2.4 times higher activity is measured in Tris-HCl buffer than in potassium phosphate buffer at the same pH values
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
62500
21000
62500
63000
64000
63000
-
gel filtration, sucrose density gradient centrifugation, determination of amino acid composition
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
trimer
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
1.6
95% loss of activity after 10 min at 4°C, reactivation within 20 min at pH 6-8
5251
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
exposure to phosphoric acid at pH 1.6 for 10 min at 4°C causes 95% or greater inactivation. Chloride ion at 50-100 mM or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. The inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8
-
irreversibly inactivated by CN- in presence of 4-hydroxy-2-oxoglutarate
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4¦C, Tris-HCl buffer, pH 7.4, 40-50% loss of activity after 1 month, more labile when frozen
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
exposure to phosphoric acid at pH 1.6 for 10 min at 4°C causes 95% or greater inactivation. The inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
preparation of (4S)-4-hydroxy-2-oxoglutarate at more than 95% enantiomeric excess by stereospecific synthesis from glyoxylate and pyruvate. Preparation of (4R)-4-hydroxy-2-oxoglutarate at 60% enantiomeric excess by selective cleavage of the (4S)-isomer of racemic 4-hydroxy-2-oxoglutarate leaving the (4R)-isomer in solution
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Nishihara, H.; Dekker, E.E.
Purification, substrate specificity and binding, beta-decarboxylase activity, and other properties of Escherichia coli 2-keto-4-hydroxyglutarate aldolase
J. Biol. Chem.
247
5079-5087
1972
Escherichia coli, Escherichia coli (P0A955)
Dekker, E.E.; Nishihara, H.; Grady, S.R.
2-keto-4-Hydroxyglutarate aldolase from Escherichia coli
Methods Enzymol.
42C
285-290
1978
Escherichia coli
-
Meloche, H.P.; Monti, C.T.; Dekker, E.E.
Enzyme stereoselectivity: the reversible reaction catalyzed by 2-keto-4-hydroxyglutarate aldolase of Escherichia coli
Biochem. Biophys. Res. Commun.
65
1033-1039
1975
Escherichia coli
Dekker, E.E.
Aldol-type reactions and 2-keto-4-hydroxyglutarate aldolase
Bioorg. Chem.
1
59-77
1977
Bos taurus, Escherichia coli
-
Wang, J.K.; Dekker, E.E.; Lewinski, N.D.; Winter, H.C.
Physical and chemical evidence for the trimeric subunit structure of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli K-12
J. Biol. Chem.
256
1793-1800
1981
Escherichia coli
Grady, S.R.; Wang, J.K.; Dekker, E.E.
Steady-state kinetics and inhibition studies of the aldol condensation reaction catalized by bovine liver and Escherichia coli 2-keto-4-hydroxyglutarate aldolase
Biochemistry
20
2497-2502
1981
Escherichia coli
Vlahos, C.J.; Dekker, E.E.
Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli
J. Biol. Chem.
265
20384-20389
1990
Escherichia coli, Escherichia coli (P0A955)
Vlahos, C.J.; Dekker, E.E.
The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase
J. Biol. Chem.
263
11683-11691
1988
Escherichia coli
Cayrol, C.; Petit, C.; Raynaud, B.; Capdevielle, J.; Guillemot, J.C.; Defais, M.
Recovery of respiration following thew SOS-response of Escherichia coli requires RecA-mediated induction of 2-keto-4-hydroxyglutarate aldolase
Proc. Natl. Acad. Sci. USA
92
11806-11809
1995
Escherichia coli
Patil, R.V.; Dekker, E.E.
Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene
J. Bacteriol.
174
102-107
1992
Escherichia coli, Escherichia coli (P0A955)
Winter, H.C.; Lewinski, N.D.; Wang, J.K.; Dekker, E.E.
Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli
Biochim. Biophys. Acta
749
52-61
1983
Escherichia coli
Vlahos, C.J.; Dekker, E.E.
Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase
J. Biol. Chem.
261
11049-11055
1986
Escherichia coli (P0A955)
Floyd, N.; Liebster, M.; Turner, N.
A simple strategy for obtaining both enantiomers from an aldolase reaction: Preparation of L- and D-4-hydroxy-2-ketoglutarate
J. Chem. Soc. Perkin Trans. I
1992
1085-1086
1992
Escherichia coli
-
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