Information on EC 4.2.1.42 - galactarate dehydratase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
4.2.1.42
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RECOMMENDED NAME
GeneOntology No.
galactarate dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
galactarate = (2R,3S)-2,3-dihydroxy-5-oxohexanedioate + H2O
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Ascorbate and aldarate metabolism
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D-galactarate degradation I
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D-galactarate degradation II
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degradation of sugar acids
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SYSTEMATIC NAME
IUBMB Comments
galactarate hydro-lyase (5-dehydro-4-deoxy-D-glucarate-forming)
The enzyme from the bacterium Escherichia coli is specific for galactarate [2], while the enzyme from Salmonella typhimurium also has activity with L-talarate (cf. EC 4.2.1.156, L-talarate dehydratase) [3]. cf. EC 4.2.1.158, galactarate dehydratase (D-threo-forming).
CAS REGISTRY NUMBER
COMMENTARY hide
37290-78-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain CR63MA
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-
Manually annotated by BRENDA team
strain CR63MA
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-
Manually annotated by BRENDA team
bifunctional L-talarate/galactarate dehydratase, activites of EC 4.2.1.42 and EC 4.2.1.156
SwissProt
Manually annotated by BRENDA team
bifunctional L-talarate/galactarate dehydratase, activites of EC 4.2.1.42 and EC 4.2.1.156
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
3-galactarate
2-dehydro-3-deoxy-D-glucarate + H2O
show the reaction diagram
D-Galactarate
5-Dehydro-4-deoxy-D-glucarate + H2O
show the reaction diagram
D-Galactarate
?
show the reaction diagram
D-galacturonate
3-deoxy-D-xylo-hexarate + 3-deoxy-D-lyxo-hexarate + H2O
show the reaction diagram
galactarate
(2R,3S)-2,3-dihydroxy-5-oxohexanedioate + H2O
show the reaction diagram
galactarate
5-dehydro-4-deoxy D-glucarate + H2O
show the reaction diagram
galactarate
5-dehydro-4-deoxy-D-glucarate + H2O
show the reaction diagram
galactarate i.e. (2R,3S,4R,5S)-2,3,4,5-tetrahydroxyhexanedioate. The enzyme also catalyzes dehydration of L-talarate. The dehydration of L-talarate is accompanied by competing epimerization to galactarate. Little epimerization to L-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with L-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with L-glucarate, a substrate for exchange of the R-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, it is concluded that Lys197 functions as the galactarate-specific base and His328 functions as the L-talarate-specific base. The epimerization of L-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His328) and epimerization (protonation on C2 by the conjugate acid of Lys197). Only galactarate and L-talarate are completely dehydrated by the enzyme. No other acid sugar results in detectable turnover
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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
3-galactarate
2-dehydro-3-deoxy-D-glucarate + H2O
show the reaction diagram
D-Galactarate
?
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
no metal requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cacodylate
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citrate
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diphosphate
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phosphate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
3-galactarate
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at pH 7.9 and 22C
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0.33 - 4.4
galactarate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12
3-galactarate
Agrobacterium tumefaciens
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at pH 7.9 and 22C
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0.0013 - 22
galactarate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
3-galactarate
Agrobacterium tumefaciens
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at pH 7.9 and 22C
197756
0.00029 - 11
galactarate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
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low activity below pH 7.0 and above pH 9.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 2 M ammonium sulfate and 100 mM Bis-Tris-HCl (pH 5.5)
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structures of the wild type enzyme complexed with L-lyxarohydroxamate, and of the K197A mutant complexed with L-glucarate. Residue Lys 197 functions as the galactarate-specific base and His 328 functions as the L-talarate-specific base
three different crystal forms are grown by hanging drop vapor diffusion at room temperature: (1) selenomethionine (SeMet)-substituted wild type enzyme, (2) wild-type enzyme complexed with Mg2+ and L-lyxarohydroxamate, and (3) the K197A mutant enzyme complexed with Mg2+ and L-glucarate
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
galactarate stabilizes
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate precipitation, column chromatography, and gel filtration
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by centrifugation, sonication, on Ni-NTA column and by gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
into a TOPO vector (pSGX3), and transformed into Escherichia coli TOP10 competent cells. Gene in the pSGX3 vector transformed into Escherichia coli strains XL1-Blue for transformation and BL21(DE3) for expression
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H45Q
has no detectable activity
R162N
retains a small amount of activity
Y164F
has no detectable activity
Y90F
is catalytically impaired. Structure of the mutant in complex with Mg2+ and galactarate has a well-defined C-terminal segment through residue 387, well-ordered electron density for galactarate, and Mg2+ ions in both metal sites for both protomers comprising the asymmetric unit
H45Q
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has no detectable activity
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R162N
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retains a small amount of activity
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Y164F
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has no detectable activity
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Y90F
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is catalytically impaired. Structure of the mutant in complex with Mg2+ and galactarate has a well-defined C-terminal segment through residue 387, well-ordered electron density for galactarate, and Mg2+ ions in both metal sites for both protomers comprising the asymmetric unit
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H328A
inactive. Mutation totally eliminates both the dehydration and epimerization activities using both L-talarate and galactarate
H328N
inactive. Mutation totally eliminates both the dehydration and epimerization activities using both L-talarate and galactarate
K197A
inactive. Mutation totally eliminates both the dehydration and epimerization activities using both L-talarate and galactarate
H328A
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inactive. Mutation totally eliminates both the dehydration and epimerization activities using both L-talarate and galactarate
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H328N
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inactive. Mutation totally eliminates both the dehydration and epimerization activities using both L-talarate and galactarate
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