Information on EC 1.1.1.271 - GDP-L-fucose synthase

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

EC NUMBER
COMMENTARY hide
1.1.1.271
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RECOMMENDED NAME
GeneOntology No.
GDP-L-fucose synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GDP-beta-L-fucose + NADP+ = GDP-4-dehydro-6-deoxy-alpha-D-mannose + NADPH + H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
GDP-L-fucose biosynthesis I (from GDP-D-mannose)
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Fructose and mannose metabolism
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Amino sugar and nucleotide sugar metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
GDP-beta-L-fucose:NADP+ 4-oxidoreductase (3,5-epimerizing)
Both human and Escherichia coli enzymes can use NADH in place of NADPH to a slight extent.
CAS REGISTRY NUMBER
COMMENTARY hide
113756-18-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
NTC 11637
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
no activity in Mycobacterium bovis
due to a natural mutation deleting the gene encoding the enzyme
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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
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-alpha-D-mannose + NADH + H+
GDP-beta-L-fucose + NAD+
show the reaction diagram
the enzyme synthesizes GDP-L-fucose from its substrate GDP-4-keto-6-deoxy-D-mannose. The reaction involves epimerization at both C-3 and C-5 followed by an NADPH-dependent reduction of the carbonyl at C-4
product identification by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy
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?
GDP-4-dehydro-6-deoxy-alpha-D-mannose + NADPH + H+
GDP-beta-L-fucose + NADP+
show the reaction diagram
GDP-4-dehydro-6-deoxy-D-mannose + NADPH
GDP-L-fucose + NADP+
show the reaction diagram
GDP-4-keto-6-deoxy-D-mannose + NADPH
GDP-6-deoxy-L-galactose + NADP+
show the reaction diagram
GDP-4-keto-6-deoxy-D-mannose + NADPH
GDP-fucose + NADP+
show the reaction diagram
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?
GDP-4-keto-6-deoxy-D-mannose + NADPH + H+
GDP-L-fucose + NADP+
show the reaction diagram
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?
GDP-6-deoxy-4-keto-mannose + NADPH
? + NADP+
show the reaction diagram
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?
additional information
?
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fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter, and GDP-fucose-synthetic enzymes, such as GDP-mannose 4,6-dehydratase, GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase, and GDP-fucose pyrophosphorylase, fucose metabolism, overview
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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
GDP-4-dehydro-6-deoxy-alpha-D-mannose + NADPH + H+
GDP-beta-L-fucose + NADP+
show the reaction diagram
D3JU54
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?
GDP-4-dehydro-6-deoxy-D-mannose + NADPH
GDP-L-fucose + NADP+
show the reaction diagram
GDP-4-keto-6-deoxy-D-mannose + NADPH
GDP-6-deoxy-L-galactose + NADP+
show the reaction diagram
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catalyzes the last two steps in the de novo synthesis of L-fucose
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?
additional information
?
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fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter, and GDP-fucose-synthetic enzymes, such as GDP-mannose 4,6-dehydratase, GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase, and GDP-fucose pyrophosphorylase, fucose metabolism, overview
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
slight activation at 5 mM
Mg2+
slight activation at 5 mM
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
complete inhibition at 5 mM
GDP
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competitive with respect to GDP-4-keto-6-deoxymannose
GDP-fucose
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competitive with respect to NADPH and GDP-4-keto-6-deoxymannose
NADP
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competitive inhibitor
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DTT
2.1fold activation
isopropyl-beta-D-thioglucopyranoside
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.047
GDP-4-dehydro-6-deoxy-alpha-D-mannose
recombinant His-tagged enzyme, pH 8.0, 37C
0.014 - 0.064
GDP-4-keto-6-deoxy-D-mannose
0.023 - 1.157
NADH
0.009 - 1.025
NADPH
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.75
GDP-4-keto-6-deoxy-D-mannose
Helicobacter pylori
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1 - 1.5
GDP-6-deoxy-4-keto-mannose
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additional information
additional information
Escherichia coli
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values for various mutants
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.061
GDP
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0.055
GDP-fucose
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0.069
NADP+
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.194
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additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.9
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7
recombinant His-tagged enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 11
activity range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
recombinant His-tagged enzyme
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15 - 55
activity range
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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increased enzyme expression compared with adjacent tissue or the tissue of healthy livers, quantitative RT-PCR expression analysis, overview
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
31500
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SDS-PAGE
35000 - 36000
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SDS-PAGE
35500
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calculated from amino acid sequence
40000
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SDS-PAGE
63100
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 40000, recombinant His-tagged enzyme, SDS-PAGE
tetramer
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two homodimers, each subunit consists of two domains, a Rossmann-fold NADPH-binding motif and a carboxyl terminal domain, quaternary structure and intermolecular contacts, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion method, complexed with NADP+
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hanging-drop vapor diffusion method, with and without NADP+
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purified recombinant enzyme, hanging drop vapor diffusion method, 5 mg/ml protein in 20 mM Tris-HCl, pH 8.0, and 100 mM NaCl, is mixed with well solution containing 100 mM Tris, pH 8.0, 30% PEG monomethyl ether 2000, X-ray diffraction structure determination and analysis at 2.37 A resolution, molecular replacement
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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wild type enzyme is stable for several weeks in phosphate buffered saline at concentrations higher than 10 mg/ml, stability of mutants differ from wild type enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 3 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by metal affinity chromatography
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homogeneity
recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography
recombinant N-terminally His6-tagged enzyme from Escherichia coli by nickel affinity chromatography and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, enzyme expression in Mycobacterium bovis leads to restoration of wild-type phenotype producing triglycosylated phenolic glycolipids instead of monoglycosylated as in the mutant, mass spectrometric lipid spectrum analysis, overview
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expressed in Escherichia coli
expressed in Saccharomyces cerevisiae
expression in Agrobacterium tumefaciens; expression in Agrobacterium tumefaciens
expression of the His-tagged enzyme in Escherichia coli strain BL21 (DE3)
gene fx, expression of the N-terminally His6-tagged enzyme in Escherichia coli
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gene wcaG, overexpression in strain BL21(DE3) together with GDP-D-mannose-4,6-dehydratase
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quantitative RT-PCR expression analysis of the enzyme in liver and hepatocarcinomas, overview
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wild-type and mutants introduced into the pT7GFS vector. Plasmid encodes for the expression of GFS bearing N-terminal hexahistidine tags, expressed in Escherichia coli BL21 (DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C109A
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lower Km than wild-type enzyme
C109S
active site mutant, produces GDP-6-deoxy-D-altrose as its major product (75%) besides GDP-L-fucose (25%) out of GDP-4-keto-6-deoxy-D-mannose
H179N
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lower Km than wild-type enzyme
H179Q
the mutant catalyzes isotope exchange into starting material indicates that its active site has not been dramatically perturbed and it is still able to bind substrate and catalyze deprotonation events
K140R
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higher Km than wild-type enzyme
K140S
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higher Km than wild-type enzyme
R187A
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lower Km than wild-type enzyme
S107A
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lower Km than wild-type enzyme
Y136E
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no enzymatic activity
C109S
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produces GDP-6-deoxy-D-altrose as its major product, thus C-3'' epimerization occurs first and premature reduction of the GDP-4-keto-6-deoxy-D-altrose intermediate becomes competitive with GDP-L-fucose production. Results in the appearance of a kinetic isotope effect when [3''-2H]-GDP-6-deoxy-4-ketomannose is used as a substrate. The mutant catalyzes a rapid wash-in of solvent derived deuterium into the C-5'' position of GDP-fucose in the presence of NADP+
H179Q
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is inactive, readily catalyzes the wash-out of deuterium from the C-3'' position of [3''-2H]-GDP-6-deoxy-4-keto-mannose
additional information
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effective GDP-L-fucose production in recombinant Escherichia coli by expression of GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxymannose 3,5-epimerase 4-reductase, method optimization at 25C and 0.1 mM isopropyl-beta-D-thioglucopyranoside, overview
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