Information on EC 3.7.1.1 - oxaloacetase

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

EC NUMBER
COMMENTARY hide
3.7.1.1
-
RECOMMENDED NAME
GeneOntology No.
oxaloacetase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
oxaloacetate + H2O = oxalate + acetate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of C-C bond
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
oxalate biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
oxaloacetate acetylhydrolase
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CAS REGISTRY NUMBER
COMMENTARY hide
9024-89-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene oah
UniProt
Manually annotated by BRENDA team
no activity in Saccharomyces cerevisiae
-
-
-
Manually annotated by BRENDA team
gene Pc22g24830
-
-
Manually annotated by BRENDA team
gene Ss-oah1
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
oxalacetate acetylhydrolase is a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2R,3S)-2,3-dimethylmalate
pyruvate + propionic acid
show the reaction diagram
-
-
-
-
?
oxaloacetate + H2O
oxalate + acetate
show the reaction diagram
additional information
?
-
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
(2R,3S)-2,3-dimethylmalate
pyruvate + propionic acid
show the reaction diagram
-
-
-
-
?
oxaloacetate + H2O
oxalate + acetate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
required, in addition to the active site metal cofactors, two additional Ca2+-binding sites mediate intermolecular interactions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3,3-difluorooxalacetate
a mechanism-based inhibitor that binds in a gem-diol form analogous to the oxalacetate intermediate/transition state, binding structure, overview
3,3-difluorooxaloacetate
3,3-difluoroxaloacetate
-
tight binding competitive inhibitor
oxalate
Phosphonopyruvate
-
-
additional information
-
(2R,3S)-isocitrate, (R)-malate, (S)-malate, and carboxyphosphoenolpyruvate are not inhibitors
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.022 - 0.14
(2R,3S)-2,3-dimethylmalate
0.53 - 0.66
(2R,3S)-2-ethyl-3-methylmalate
0.004 - 0.021
Mn2+
0.027 - 20.14
oxaloacetate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0249 - 0.074
(2R,3S)-2,3-dimethylmalate
5.4 - 8.4
(2R,3S)-2-ethyl-3-methylmalate
0.00001 - 50
oxaloacetate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 3000000
oxaloacetate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000068
3,3-difluorooxaloacetate
-
-
0.0025
3,3-difluoroxaloacetate
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in 50 mM K+-HEPES (pH 7.5 and 25C)
1.6
oxalate
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in 50 mM K+-HEPES (pH 7.5 and 25C)
0.0024
Phosphonopyruvate
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in 50 mM K+-HEPES (pH 7.5 and 25C)
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.3
-
specific activity determined for fungus grown for 4 days on ammonium tartrate-containing medium
0.62
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cell extract
4.6
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purification step ammonium sulfate precipitation
15
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purification step DEAE-cellulose column
20
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purification step butyl-Sepharose column
58
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partially purified enzyme
additional information
-
relative activity, supernatant fraction obtained from centrifugation at 3000 g 100%, supernatant at 20000 g 70%, pellet 10% activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
-
in MES, piperazine-N,N'-bis(2-ethansulfonic acid), Tris and triethanolamine
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34360
-
theoretical molecular mass
35000
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determined by SDS-PAGE
100000
-
determined by gel filtration, indicative of a homotrimeric quaternary structure
110000
-
gel filtration
250000
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gel filtration
360000 - 440000
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two major bands, native PAGE
420000
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zonal centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
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4 * 31986, mass spectrometry; 4 * 32000, SDS-PAGE; 4 * 32117, calculated from amino acid sequence
oligomer
-
10-12 * 39000, SDS-PAGE
tetramer
OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)8 barrel fold and each pair swapping the 8th alpha-helix
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DMML in complex with Mg2+ and in complex with Mg2+ and 3,3-difluorooxalacetate, hanging drop vapor diffusion method, using 0.2 M potassium thiocyanate, 0.1 M bis-Tris propane (pH 7.5), and 20k polyethylene glycol 3350, or using 14k polyethylene glycol 6000 and 0.1 M MES (pH 6.5)
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OAH alone, in complex with a inhibitor 3,3-difluorooxalacetate, and in complex with reaction product oxalate, soaking of ligand-free OAH crystals in solution containing 0.2 M MnCl2 or 0.2 M MgCl2, 10 mM CaCl2, 2 mM 3,3-difluorooxalacetate, 0.1 M Na-HEPES, pH 7.5, and 30% v/v PEG 400 for 5-10 min yielded the desired enzyme inhibitor complex, X-ray diffraction structure determination and analysis at resolution limit of 1.30, 1.55, and 1.65 A , respectively, molecular replacement
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.8
-
loss of activity below
210610
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
unstable if Mn2+ concentration is low
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18C, lyophilized, 3 months
-
4C, 50 mM Tris-HCL, pH 7.5, 2 mM MnCl2, 20 mM DTT 5% sucrose, 7 days
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by ammonium sulfate-induced protein precipitation, and by using of a DEAE-cellulose and a butyl-Sepharose column
-
Phenyl Sepharose, Q-Sepharose
-
proteins are purified to homogeneity
Q-Sepharose column chromatography, ammonium sulfate precipitation, butyl Sepharose column chromatography, and phenyl Sepharose column chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned by inverse PCR into Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
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gene An07g08390, DNA and amino acid sequence determination and analysis; gene An15g07720, DNA and amino acid sequence determination and analysis; gene oah, cloned from strain WU-2223L, recombinant overexpression in Aspergillus strain WU-2223L, construction of plasmids pDOAHPTR-1 and pNANOAH-1 for enzyme overexpression in strainWU-2223L
gene gene Ss-oah1, DNA and amino acid sequence determination and analysis, PCR-based genome walking
gene oahA, phylogenetic tree, recombinant expression in Escherichia coli strain BL21(DE3)
gene Pc22g24830, DNA and amino acid sequence determination and analysis, cloning in Escherichia coli strain DH5alpha, functional expression in Saccharomyces cerevisiae strain IME043, IME046, and IME047
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into the vector pET-3c
into the vector pGEM-T Easy for sequencing
into the vector pGEM-T Easy for sequencing, and into pET-3c for expression in Escherichia coli BL21DE3 cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C124A
-
the mutant shows decreased kcat compared to the wild type enzyme
C124S
-
the mutant shows decreased kcat compared to the wild type enzyme
D59A
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the mutant shows decreased kcat compared to the wild type enzyme
D59S
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the mutant shows decreased kcat compared to the wild type enzyme
P240S
-
the mutant shows decreased kcat compared to the wild type enzyme
P240T
-
the mutant shows decreased kcat compared to the wild type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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oxalate production by Moniliophthora perniciosa may play a role in the Witches' Broom disease pathogenesis mechanism
food industry