Information on EC 4.1.1.85 - 3-dehydro-L-gulonate-6-phosphate decarboxylase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY hide
4.1.1.85
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RECOMMENDED NAME
GeneOntology No.
3-dehydro-L-gulonate-6-phosphate decarboxylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-dehydro-L-gulonate 6-phosphate + H+ = L-xylulose 5-phosphate + CO2
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Ascorbate and aldarate metabolism
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ascorbate metabolism
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L-ascorbate degradation I (bacterial, anaerobic)
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L-ascorbate degradation II (bacterial, aerobic)
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Metabolic pathways
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Microbial metabolism in diverse environments
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Pentose and glucuronate interconversions
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SYSTEMATIC NAME
IUBMB Comments
3-dehydro-L-gulonate-6-phosphate carboxy-lyase (L-xylulose-5-phosphate-forming)
Requires Mg2+. Along with EC 5.1.3.22, L-ribulose-5-phosphate 3-epimerase, this enzyme is involved in a pathway for the utilization of L-ascorbate by Escherichia coli.
CAS REGISTRY NUMBER
COMMENTARY hide
406722-60-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain UA159
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Manually annotated by BRENDA team
strain UA159
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-dehydro-L-gulonate 6-phosphate + H+
L-xylulose 5-phosphate + CO2
show the reaction diagram
additional information
?
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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-dehydro-L-gulonate 6-phosphate + H+
L-xylulose 5-phosphate + CO2
show the reaction diagram
additional information
?
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enzyme catalyzes one step in the L-ascorbate utilization pathway, L-ascorbate is converted to L-xylulose, overview
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
L-gulonate 6-phosphate
binding structure involving Glu33 and Asp62 and Mg2+
L-xylitol 5-phosphate
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binding structure
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15 - 5.4
3-dehydro-L-gulonate 6-phosphate
additional information
additional information
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analysis of kinetics and activity of the mutant enzymes, overview
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.19 - 64
3-dehydro-L-gulonate 6-phosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
additional information
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(beta/alpha)8-barrel enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme complexed with L-gulonate 6-phosphate, L-threonohydroxamate 4-phosphate, and L-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product L-xylulose 5-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM Bis-Tris propane, pH 7.0, and 5 mM MgCl2, with 25 mM ligand, X-ray diffraction structure determination and analysis at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively
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purified recombinant mutant enzymes K64A, H136A, E112Q, and E112Q/H136A, in complex with reaction intermediate analogue L-threonohydroxamate 4-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM Bis-Tris propane, pH 7.0, 5 mM MgCl2, and 25 mM L-threonohydroxamate 4-phosphate, X-ray diffraction structure determination and analysis at 1.7, 1.9, 1.8, and 1.9 A resolution, respectively
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purified recombinant wild-type and selenomethionine-labeled enzymes, free or complexed with inhibitor L-gulonate 6-phosphate, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl, room temperature, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 18% monomethyl PEG 5000, 50 mM NaH2PO4, 50 mM K2HPO4, and 50 mM Bis-Tris propane, pH 7.0, with or without 20 mM inhibitor L-gulonate 6-phosphate, crystals appear a few days after microseeding, growing for 1 week, X-ray diffraction structure determination and analysis at 2.0 A resolution
purified recombinant wild-type and mutant enzymes E112D/R139V, E112D/T169A, and E112D/R139V/T169A, 15 mg/ml protein in 50 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl, micro-batch method, 0.01 ml protein solution mixed with equal volume of crystallization solution containing 16% monomethyl PEG 5000, 100 mM Bis-Tris propane, pH 7.0, 5 mM MgCl2, and 25 mM L-xylulose 5-phosphate or D-ribulose 5-phosphate, cryoprotection of crystals in 15% monomethylPEG 5000, 100 mM PIPES, pH 7.0, 15% ethylene glycol, 200 mM NaCl, and 50 mM L-threonohydroxamate 4-phosphate or 50 mM D-ribulose 5-phosphate, X-ray diffraction structure determination and analysis at 1.6-1.8 A resolution, molecular replacement
at 289 K using the hanging-drop vapor-diffusion technique. Apoenzyme obtained in two different space groups P212121 (without Mg2+) and I422 (in complex with Mg2+) and in the absence and presence of the product analog D-ribulose 5-phosphate. An 8 A alphaB-helix movement and other structural rearrangements around the active site between the apostructures and product analog bound structure. Conformational flexibility around the active site for substrate/product binding. Especially the alphaB-helix (residues 144-149) and the side chains of the conserved Arg144 and Arg197 exhibit openclosed conformational changes between the apo-structure and complex structure with ligand bound at the active site
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
on Ni2+-chelating affinity column and by gel filtration
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recombinant His-tagged UlaD from strain BL21(DE3), the His-tag is removed by thrombin
recombinant N-terminally His10-tagged SgbH from strain BL21(DE3) to homogeneity, the His-tag is removed by thrombin
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of mutant enzymes in strain BLR(DE3)recA-strain
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expression of N-terminally His10-tagged wild-type and mutant enzymes in enzyme-deficient strain BLR(DE3)
gene sgbH, expression in strain BL21(DE3) as N-terminally His10-tagged protein
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gene ulaD, expression as His-tagged protein in strain BL21(DE3), and as selenomethionine-labeled enzyme
into pET-28a vector containing an N-terminal His6-tag and expressed in Escherichia coli strain BL21 (DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E112Q
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site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry
E112Q/H136A
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site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry
H136A
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site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry
K64A
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site-directed mutagenesis, crystal structure determination and analysis, the mutant enzyme shows altered reaction intermediate binding at the active site, reaction mechanism, and stereochemistry
D67A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
D67A/H136A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
D67N
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
D67N/H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
E112A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
E112A/H136A
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
E112A/H136A/R139V
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
E112D/R139V
site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens D-arabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-L-gulonate 6-phosphate decarboxylase performing th Mg2+-dependent aldol condensation between formaldehyde and D-ribulose 5-phosphate with highly increased activity compared to the wild-type enzyme
E112D/R139V/T169A
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site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens D-arabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-L-gulonate 6-phosphate decarboxylase performing th Mg2+-dependent aldol condensation between formaldehyde and D-ribulose 5-phosphate with highly increased activity compared to the wild-type enzyme
E112D/R139V/T169A/R192A
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site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens D-arabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-L-gulonate 6-phosphate decarboxylase performing th Mg2+-dependent aldol condensation between formaldehyde and D-ribulose 5-phosphate with highly increased activity compared to the wild-type enzyme
E112D/T169A
site-directed mutagenesis, mutations alter the Escherichia coli residues to those of Methylomonas aminofaciens D-arabino-hex-3-ulose 6-phosphate synthase, a homologous enzyme, also altering the enzyme activity of the 3-keto-L-gulonate 6-phosphate decarboxylase performing th Mg2+-dependent aldol condensation between formaldehyde and D-ribulose 5-phosphate with highly increased activity compared to the wild-type enzyme
E112Q
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
E112Q/H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
E112Q/H136A/R139V
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
E33K
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site-directed mutagenesis, inactive mutant
H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
H136A/R139V
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
H136N
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
H136Q
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
K64A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
K64A/E112A/H136A/R139V
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
K64A/E112Q/H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
K64A/H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
K64A/R139V
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
K64R
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site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
K64R/H136A
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site-directed mutagenesis, reduced activity compared to the wild-type enzyme
R139V
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site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
Show AA Sequence (1250 entries)
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