Information on EC 4.2.1.47 - GDP-mannose 4,6-dehydratase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.1.47
-
RECOMMENDED NAME
GeneOntology No.
GDP-mannose 4,6-dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GDP-alpha-D-mannose = GDP-4-dehydro-alpha-D-rhamnose + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
elimination
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
-
-
Fructose and mannose metabolism
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GDP-6-deoxy-D-talose biosynthesis
-
-
GDP-D-perosamine biosynthesis
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GDP-D-rhamnose biosynthesis
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-
GDP-L-colitose biosynthesis
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GDP-L-fucose biosynthesis I (from GDP-D-mannose)
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GDP-mycosamine biosynthesis
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Metabolic pathways
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d-mannose degradation
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SYSTEMATIC NAME
IUBMB Comments
GDP-mannose 4,6-hydro-lyase (GDP-4-dehydro-6-deoxy-D-mannose-forming)
The bacterial enzyme requires bound NAD+. This enzyme forms the first step in the biosynthesis of GDP-alpha-D-rhamnose and GDP-beta-L-fucose. In Aneurinibacillus thermoaerophilus L420-91T, this enzyme acts as a bifunctional enzyme, catalysing the above reaction as well as the reaction catalysed by EC 1.1.1.281, GDP-4-dehydro-6-deoxy-D-mannose reductase [5]. Belongs to the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr and Ser/Thr residues [6].
CAS REGISTRY NUMBER
COMMENTARY hide
37211-59-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
-
Manually annotated by BRENDA team
ATCC 29148D-5
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-
Manually annotated by BRENDA team
bovine
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain K12
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
expression of GMD is higher in macrophages than in endothelial cells; high expression
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-
Manually annotated by BRENDA team
polychaete worm
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-
Manually annotated by BRENDA team
Paramecium bursaria chlorella virus
-
-
-
Manually annotated by BRENDA team
Paramecium bursaria Chlorella virus-1
-
-
-
Manually annotated by BRENDA team
Phaseolus aureus, mung bean
-
-
Manually annotated by BRENDA team
expression of GMD is higher in macrophages than in endothelial cells; expression of GMD was higher in macrophages than in endothelial cells; high expression
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-
Manually annotated by BRENDA team
brewer's yeast
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-
Manually annotated by BRENDA team
pig
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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mutations in this enzyme participate in the progression of colorectal cancer
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
GDP-4-dehydro-6-deoxy-D-mannose
GDP-D-rhamnose
show the reaction diagram
i.e., GDP-6-deoxy-D-lyxo-hexos-4-ulose
-
-
?
GDP-alpha-D-mannose
GDP-4-dehydro-alpha-D-rhamnose + H2O
show the reaction diagram
GDP-D-mannose
GDP-4-dehydro-6-deoxy-D-mannose + H2O
show the reaction diagram
GDP-mannose
GDP-4-dehydro-6-deoxy-D-mannose + H2O
show the reaction diagram
GDP-mannose
GDP-4-keto-6-deoxy mannose + H2O
show the reaction diagram
-
-
-
?
GDPmannose
?
show the reaction diagram
GDPmannose
GDP-4-dehydro-6-deoxy-D-mannose + H2O
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
GDP-alpha-D-mannose
GDP-4-dehydro-alpha-D-rhamnose + H2O
show the reaction diagram
GDP-mannose
GDP-4-dehydro-6-deoxy-D-mannose + H2O
show the reaction diagram
-
key regulatory enzyme controlling alginate biosynthesis in mucoid strain of pseudomonas aeruginosa
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-
?
GDP-mannose
GDP-4-keto-6-deoxy mannose + H2O
show the reaction diagram
D3JU53
-
-
-
?
GDPmannose
?
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
activates
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
-
50% inhibition
Cu2+
5 mM, strong inhibition; strong inhibition at 5 mM
Fe2+
5 mM, strong inhibition; strong inhibition at 5 mM
GDP-alpha-D-mannose
substrate inhibition
GDP-D-glucose
GDP-fucose
-
competitive inhibition
GDP-L-fucose
GDP-L-glucose
competitive
GMP
-
90% inhibition
ITP
-
90% inhibition
Mg2+
high concentrations of MgCl2 (25 mM) have an inhibitory effect on enzyme activity
Mn2+
-
90% inhibition
NADP+
Ni2+
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90% inhibition
p-Chloromercuriphenylsulfonate
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-
p-hydroxymercuribenzoate
-
-
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dithiothreitol
enhances GMD activity by 2.3fold
DTT
enhances GMD activity by 2.3fold
NADPH
Pyridine nucleotides
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-
-
additional information
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specific activity activity of GDP-mannose dehydrogense increases with dissolved oxygen tension of up to 25% of air saturation and maintains the maximal value up to at least 70% of air saturation
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.024
GDP-alpha-D-mannose
at pH 7.4 and 25C, in the presence of 1 mol MgCl2 per mol of protein
0.0035 - 1.02
GDP-mannose
0.003 - 0.55
GDPmannose
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1
GDP-alpha-D-mannose
Burkholderia cepacia
B4EFL2
at pH 7.4 and 25C, in the presence of 1 mol MgCl2 per mol of protein
0.0015 - 8.82
GDP-mannose
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.977
GDP-alpha-D-mannose
Burkholderia cepacia
B4EFL2
at pH 7.4 and 25C, in the presence of 1 mol MgCl2 per mol of protein
3633
630
GDP-mannose
Pseudomonas aeruginosa
Q51366
pH 7.5, 37C
366
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.913
GDP-alpha-D-mannose
at pH 7.4 and 25C, in the presence of 1 mol MgCl2 per mol of protein
0.084
GDP-D-glucose
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-
0.0789
GDP-fucose
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pH 7, 37C
0.0113
GDP-L-fucose
pH 8
0.002859
GDP-mannose
pH 7.5, 37C
0.186
GTP
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.036
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-
0.11
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-
0.48
Paramecium bursaria Chlorella virus-1
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2.3
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determined with fusion protein
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.1
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-
9
; recombinant enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
pH 5: 45% of maximal activity, pH 11: 75% of maximal activity
5 - 9.2
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5.5 - 8
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6.5 - 8
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 55
25C: about 60% of maximal activity, 55C: about 70% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
-
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
additional information
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including cultured cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38700
isoenzyme S-GMD, SDS-PAGE
40000
-
x * 40000, SDS-PAGE
40200
isoenzyme M-GMD, SDS-PAGE
41200
3 * 45000, recombinant His-tagged GMD, SDS-PAGE, 3 * 41200, about, sequence calculation
41500
-
6 * 41500, SDS-PAGE
45000
3 * 45000, recombinant His-tagged GMD, SDS-PAGE, 3 * 41200, about, sequence calculation; 3 * 45000, SDS-PAGE
80000
isoenzyme M-GMD, dimeric form, gel filtration
87000
Paramecium bursaria Chlorella virus-1
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gel filtration
110000
-
gel filtration
120000
-
sedimentation equilibrium centrifugation
140000
gel filtration; recombinant His-tagged GMD, native PAGE
162000
gel filtration
250000
-
gel filtration
251000
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sedimentation equilibrium centrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
tetramer
trimer
3 * 45000, recombinant His-tagged GMD, SDS-PAGE, 3 * 41200, about, sequence calculation; 3 * 45000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, complexed with NADPH, GDP and GDP-D-rhamnose
hanging drop vapor diffusion method
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crystal structure at 3.8 A resolution
Paramecium bursaria chlorella virus
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complexed with NADPH and GDP, hanging drop vapor diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
unstable to freezing except at high sucrose concentration
4
-
stable for 1 day, 50% loss of activity
25
His6-GMD loses activity slowly in the course of enzyme-substrate incubations, particularly at 37C, but 4,6-dehydratase activity is still detectable after incubation for 16 h at 25C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
NADPH is essential for stabilization and function of the enzyme
very unstable
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is not very sensitive to oxidative inactivation. Specific activity activity of GDP-mannose dehydrogense increases with dissolved oxygen tension of up to 25% of air saturation and maintains the maximal value up to at least 70% of air saturation
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665895
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, 15 months, 40% loss of activity
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-15C, 70% ammonium sulfate solution, several weeks
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-20C, 2 M sucrose, 4 months
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-80C, 25% glycerol, 1 year
4C, presence of reducing agents, 36 h, 50% loss of activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; GSH-Sepharose 4B column chromatography
by ultrasonication, centrifugation, on Ni2+-nitrilotriacetic acid resin and by anion exchange chromatography, to greater than 95% purity
Hi-Trap chelating column chromatography
only isoenzyme L-GMD and isoenzyme M-GMD are soluble, isoenzyme S-GMD is inactive and forms a precipitate
recombinant His-tagged GMD from Escherichia coli by nickel affinity chromatography; to near homogeneity by nickel ion affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as fusion protein
-
coexpression with GDP-4-keto-6-deioxymannose-3,5-epimerase-4-reductase, which is required for enzymatic activity after expresseion in Saccharomyces cerevisiae
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expressed in Corynebacterium glutamicum strain ATCC 13032
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expressed in Escherichia coli
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expressed in Escherichia coli as glutathione S-transferase fusion protein; expression in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
expression in Agrobacterium tumefaciens; expression in Agrobacterium tumefaciens
expression in Escherichia coli
GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxymannose 3, 5-epimerase 4-reductase, the two crucial enzymes for the de novo GDP-L-fucose biosynthesis, are overexpressed in recombinant Escherichia coli by constructing inducible overexpression vectors. Optimum expression conditions in recombinant Escherichia coli BL21(DE3) are 25C and 0.1 mM isopropyl-beta-D-thioglucopyranoside. Maximum GDP-L-fucose concentration of 38.9 mg/l is obtained in a glucose-limited fed-batch cultivation, and it is enhanced further by coexpression of NADPH-regenerating glucose-6-phosphate dehydrogenase encoded by the zwf gene to achieve 55.2 mg/l GDP-L-fucose under the same cultivation condition
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GMD DNA and amino acid sequence determination and analysis
GMD sequence determination and analysis, sequence comparison and phylogenetic tree, functional overexpression of soluble GMD in Escherichia coli strain BL21(DE3) from an IPTG-inducible expression vector, optimum expression conditions are 30C and 0.1 mM IPTG
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His6-GMD expressed from the pQE-30-gmd vector
into pET28a+ to construct pLW1534, expressed in Escherichia coli BL21 as His-tagged fusion protein; recombinant expression of His-tagged GMD in Escherichia coli
L-GMD, S-GMD and M-GMD, expressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K161A
-
3000fold reduced kcat
T133V
-
3000fold reduced kcat
Y157F
-
3000fold reduced kcat
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants
synthesis
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