Information on EC 1.1.1.22 - UDP-glucose 6-dehydrogenase

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

EC NUMBER
COMMENTARY hide
1.1.1.22
-
RECOMMENDED NAME
GeneOntology No.
UDP-glucose 6-dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-glucose + 2 NAD+ + H2O = UDP-alpha-D-glucuronate + 2 NADH + 2 H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Amino sugar and nucleotide sugar metabolism
-
-
Ascorbate and aldarate metabolism
-
-
Metabolic pathways
-
-
non-pathway related
-
-
Pentose and glucuronate interconversions
-
-
Starch and sucrose metabolism
-
-
UDP-alpha-D-glucuronate biosynthesis (from UDP-glucose)
-
-
SYSTEMATIC NAME
IUBMB Comments
UDP-alpha-D-glucose:NAD+ 6-oxidoreductase
Also acts on UDP-alpha-D-2-deoxyglucose.
CAS REGISTRY NUMBER
COMMENTARY hide
9028-26-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene bceC
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
cultivar NVS or K326
SwissProt
Manually annotated by BRENDA team
two-dimensional IEF SDS-PAGE showed several isoforms of the purified enzyme
-
-
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
pea
-
-
Manually annotated by BRENDA team
induction by short-term feeding with sucrose, sorbitol, ethylene glycol or light exposure
SwissProt
Manually annotated by BRENDA team
gene ugd
UniProt
Manually annotated by BRENDA team
-
UniProt
Manually annotated by BRENDA team
Saccharum spp.
hybrid
-
-
Manually annotated by BRENDA team
gene ugdG
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3-OH-benzopyrene + NAD+
?
show the reaction diagram
-
-
-
-
?
5-azido-UDP-glucose + NAD+
5-azido-UDP-glucuronate + NADH + H+
show the reaction diagram
-
-
-
-
?
5-fluorouracil + NAD+
?
show the reaction diagram
-
-
-
-
?
6-azauracil + NAD+
?
show the reaction diagram
-
-
-
-
?
CDP-glucose + NAD+ + H2O
CDP-glucuronate + NADH
show the reaction diagram
CTP-glucose + NAD+
CTP-glucuronate + NADH
show the reaction diagram
Saccharum spp.
-
8% of activity with UDP-glucose
-
-
ir
dTDP-glucose + NAD+ + H2O
dTDP-glucuronate + NADH
show the reaction diagram
-
reaction rate is 16.7% of that with UDPglucose
-
-
?
TDP-glucose + NAD+
TDP-glucuronate + NADH
show the reaction diagram
Saccharum spp.
-
2% of activity with UDP-glucose
-
-
ir
TDP-glucose + NAD+ + H2O
TDP-glucuronate + NADH
show the reaction diagram
UDP-2-deoxy-D-glucose + NAD+ + H2O
UDP-2-deoxy-D-glucuronate + NADH
show the reaction diagram
-
-
-
-
?
UDP-alpha-D-glucose + 2 NAD+ + H2O
UDP-alpha-D-glucuronate + 2 NADH + 2 H+
show the reaction diagram
UDP-alpha-D-mannose + 2 NAD+ + H2O
UDP-alpha-D-mannuronate + 2 NADH + 2 H+
show the reaction diagram
UDP-D-galactose + 2 NAD+ + H2O
UDP-alpha-D-galacturonate + 2 NADH + 2 H+
show the reaction diagram
UDP-galactose + NAD+ + H2O
UDP-galacturonate + NADH
show the reaction diagram
UDP-glucose + 2 NAD+ + H2O
UDP-glucuronate + 2 NADH + 2 H+
show the reaction diagram
UDP-glucose + 2 NAD+ + H2O
UDP-glucuronic acid + 2 NADH + 2 H+
show the reaction diagram
UDP-glucose + 3-acetylpyridine adenine dinucleotide + H2O
UDP-glucuronate + ?
show the reaction diagram
UDP-glucose + 3-pyridinealdehyde adenine dinucleotide
UDP-glucuronate
show the reaction diagram
-
-
-
-
?
UDP-glucose + deamino adenine dinucleotide + H2O
UDP-glucuronate + ?
show the reaction diagram
UDP-glucose + nicotinamide hypoxanthine dinucleotide + H2O
UDP-glucuronate + ?
show the reaction diagram
-
-
-
-
?
UDP-glucose + thionicotinamide adenine dinucleotide + H2O
UDP-glucuronate + ?
show the reaction diagram
UDP-N-acetylglucosamine + NAD+ + H2O
? + NADH
show the reaction diagram
UDPglucose + NAD+ + H2O
UDPglucuronate + NADH
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
UDP-alpha-D-glucose + 2 NAD+ + H2O
UDP-alpha-D-glucuronate + 2 NADH + 2 H+
show the reaction diagram
UDP-glucose + 2 NAD+ + H2O
UDP-glucuronate + 2 NADH + 2 H+
show the reaction diagram
UDP-glucose + 2 NAD+ + H2O
UDP-glucuronic acid + 2 NADH + 2 H+
show the reaction diagram
UDPglucose + NAD+ + H2O
UDPglucuronate + NADH
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-acetylpyridine adenine dinucleotide
beta-NAD+
-
-
additional information
-
no constantly bound chromophoric cofactors, i.e. NAD+, required
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
is able to function in a variety of salt concentrations, enzymatic activity improves as salinity decreases. At 4 M NaCl concentration the enzyme was able to produce 870 nM NADH, while at 0.5 M NaCl, over 1.5 mM NADH is produced
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Ca2+
84% residual activity at 1 mM
EDTA
83% residual activity at 1 mM
gallic acid
-
is a non-competitive inhibitor with respect to UDP-glucose and NAD+. It decreases specific activities of UGDH, but does not affect UGDH protein expression, thus UGDH activity is inhibited by polyphenols at the post-translational level. Gallic acid exerts strong antiproliferative activity in breast cancer cells. Heat inactivation of UGDH is accelerated to a greater degree by quercetin than by gallic acid. In the presence of gallic acid, the activity remaining after 30 min is 55% that of control
Mn2+
84% residual activity at 1 mM
NH2OH
-
deactivation
p-chloromercuribenzoate
piperine
quercetin
-
shows a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, decreases specific activities of UGDH, but does not affect UGDH protein expression, thus UGDH activity is inhibited by polyphenols at the post-translational level. Quercetin exerts strong antiproliferative activity in breast cancer cells. Heat inactivation of UGDH is accelerated to a greater degree by quercetin than by gallic acid. In the presence of quercetin, the activity remaining after 30 min is 20% that of control
thiol group modifying reagents
-
-
-
UDP
-
0.5 mM, partial inhibition
UDP-alpha-D-glucuronate
-
0.05 mM, partial inhibition
UDP-alpha-D-xylose
-
competitive. The DELTA132 deletion mutant and the UDP-alpha-D-xylose-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states
UDP-D-galactose
-
slight
UDP-D-xylose
UDP-galactose
-
0.5 mM, partial inhibition
UDP-glucuronate
Saccharum spp.
-
competitive
UDP-glucuronic acid
product inhibition, 44% residual activity at 1 mM; product inhibition, 79% residual activity at 1 mM
UDP-xylose
UDParabinose
-
-
UDPgalacturonic acid
-
-
UDPglucuronate
UDPxylose
Uridine 5'-diphosphate chloroacetol
-
alkylates a thiol group of cysteine in the catalytic centre via being specifically bound instead of the substrate
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
17beta-estradiol
-
upregulation of UDP-glucose dehydrogenase at mRNA, protein and activity level in articular chondrocyte
3-pyridinealdehyde adenine dinucleotide
-
can replace NAD+
Deamino adenine dinucleotide
lactose
-
UDP-GlcDH activities in extracts of Lactococcus lactis strain NFHA01 induced with both 0.5% and 2% lactose are significantly higher than that of the same strain without induction. UDP-GlcDH activity of NFHA01 induced with 2% lactose is about 110% higher than that of 0.5% lactose induction
nicotinamide hypoxanthine dinucleotide
-
can replace NAD+
nisin
-
induces the expression of szHasA together with szHasB
TGF-beta
-
enhances enzyme activity in articular chondrocyte
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Thionicotinamide adenine dinucleotide
additional information
-
fetal calf serum stimulates enzyme activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05617
5-azido-UDP-glucose
-
22C, pH 8.7
0.058
aldehyde intermediate
-
-
-
0.006 - 6.3
NAD+
0.017 - 0.98
UDP
0.016 - 2.21
UDP-alpha-D-glucose
0.0092 - 1.5
UDP-glucose
0.015 - 8.4
UDPglucose
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.9
aldehyde intermediate
Streptococcus pyogenes
-
-
-
0.02 - 7.6
NAD+
97 - 105
UDP
0.0014 - 6.9
UDP-alpha-D-glucose
0.127 - 8.73
UDP-glucose
0.117
UDPglucose
Streptococcus pyogenes
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.014 - 1.2
NAD+
0.025 - 24
UDP-alpha-D-glucose
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.744 - 1.804
gallic acid
0.0005
p-chloromercuribenzoate
-
pH 8.7, 25C
0.07 - 0.083
quercetin
0.292
UDP-glucuronate
Saccharum spp.
-
pH 8.4, 25C
0.017 - 0.22
UDP-xylose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000031
-
mutant D280N, pH 7.4, 22C
0.00022
-
mutant K276A, pH 7.4, 22C
0.0004
-
mutant K220H, pH 7.4, 22C
0.00041
-
mutant K220R, pH 7.4, 22C
0.00051
-
lysates of cells treated with 0.3 mM gallic acid
0.00061
-
lysates of cells treated with 0.3 mM quercetin
0.0015
-
control lysates
0.00247
-
mutant K279A, pH 7.4, 22C
0.0027
-
mutant K276S, pH 7.4, 22C
0.0029
-
mutant K220A, pH 7.4, 22C
0.41
-
mutant D280E, pH 7.4, 22C
0.69
-
wild-type, 22C, pH 7.4
0.74
-
mutant K339A, pH 7.4, 22C
1.5
-
recombinant and purified
2.17
Saccharum spp.
-
pH 8.4, 25C
27.17
-
purified 390fold
342.9
-
pH 8.8, 25C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.4 - 8.8
-
-
8.4
Saccharum spp.
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 11
no enzymic activity beyond
6.3 - 8.6
-
at pH 6.3 and 8.6 about 50% of activity maximum
7
37% of maximum activity
7 - 9.5
-
-
7
-
46% of maximum acitivity
7.5 - 9.4
-
at pH 7.5 and 9.4 about 50% of activity maximum
7.8 - 9.4
-
at pH 7.8 and 9.4 about 50% of activity maximum
8.8
-
80-83% of maximum activity
9.5 - 11
-
pH 9.5: about 40% of maximal activity, pH 11.0: about 10% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 45
-
-
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
significant decrease in activity below
30
-
90% of maximum activity
40
70% of maximum activity
50
no enzymic activity above
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
silencing of UGDH decreases glycosaminoglycan synthesis causing severe embryonic malformations because of defective gastrulation process. Overexpression of the enzyme from Xenopus laevis in human smooth muscle cells increases the accumulation of hyaluronan
Manually annotated by BRENDA team
-
enzyme activity depends on growth phase of the culture
Manually annotated by BRENDA team
the abundance of enzyme mRNA in pistils is more than 3fold greater than that in other parts, and the abundance in stamens and calyx tubes is relatively high compared with that in sepals and petals
Manually annotated by BRENDA team
changes in the mRNA level during peach fruit development correspond to changes in the amount of cell wall material and the cell wall uronic acid content. These are greater in the fruits of the commercial cultivars compared with the Japanese native peach cultivars, and the expression of enzyme is higher in the fruits of the commercial cultivars
Manually annotated by BRENDA team
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prostate tumor cell line derived from PC-3 cells
Manually annotated by BRENDA team
-
high expression level
Manually annotated by BRENDA team
-
increased UGDH expression in cancerous acini and decreased expression in normal-appearing acini of the same prostate relative to acini of non-cancerous prostates
Manually annotated by BRENDA team
Saccharum spp.
-
-
Manually annotated by BRENDA team
enzyme activity increases dramatically in a special subset of vulval cells during vulval morphogenesis
Manually annotated by BRENDA team
-
siRNA for the human enzyme is put into a pRNA-U6.1/Neo vector and chemically transfected into bresat cancer cells. The UGDH siRNA plasmid then knocks down UGDH expression in ZR-75-1 cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Porphyromonas gingivalis (strain ATCC BAA-308 / W83)
Pyrobaculum islandicum (strain DSM 4184 / JCM 9189 / GEO3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
44000
-
gel filtration
45500
-
electrospray mass spectrometry
52000
-
gel filtation
55000
-
gel filtration
57000
-
gel filtration, minor part of wild-type
72000
-
gel filtration
86000
-
gel filtration
104000
gel filtration
110000
-
gel filtration, minor part of wild-type, mutant A222Q/S233G
117000
gel filtration
300000
305000
-
equilibrium measurement under native conditions
340000
345000
gel filtration, dynamic light scattering
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homodimer
recombinant form of Ugd(BCAL2946); recombinant form of Ugd(BCAM0855)
monomer
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His6-tagged BceC, 0.001 ml of protein solution containing 5 mg/ml protein in 25 mM Tris-HCl, pH 8.3, 50 mM NaCl, 2.5 mM DTT, 0.25 mM UDP-GlcA, and 0.5 mM NAD+, is mixed with 0.001 ml of precipitant solution containing 200 mM ammonium sulfate, 100 mM sodium acetate, pH 4.5, 11% w/v PEG 4000, and 50 mM NaF, method optimiization, X-ray diffraction structure determination and analysis at 2.09 A resolution, molecular replacement
-
to 1.75 A resolution
2.3 A resolution crystal structure of the deletion construct DELTA132 reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. The open conformation stabilizes the deletion construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDP-alpha-D-xylose-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. The DELTA132 and the UDP-alpha-D-xylose-inhibited structures have similar hexamer-building interfaces
-
alternate crystal structure of human enzyme in complex with UDP-glucose at 2.8 A resolution. The substrate-bound protein complex consists of the open homohexamer. In all subunits of the open structure, residue Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water molecule bound to Asp280 while protecting the reaction intermediates
-
crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data are collected to a resolution of 2.8 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A and alpha = beta = gamma = 90.0
-
in mutant E161Q, the hydrolysis step becomes completely rate-limiting so that a thioester enzyme intermediate accumulates at steady state. Crystallization of mutant E161Q in the presence of 5 mM UDP-glucose and 2 mM NAD results in trapping a thiohemiacetal enzyme intermediate. Residue Cys276 is covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction
-
mutant K94E, to 2.08 A resolution. Cofactor binding triggers the formation of the 32 symmetry hexamer, but substrate UDP-alpha-D-glucose is needed for the stability of the complex. Loop88-110 is the cofactor-responsive allosteric switch that drives hexamer formation, loop88-110 directly links cofactor binding to the stability of the hexamer-building interface. In the interface, loop88-110 packs against the Thr131-loop/alpha6 helix, the allosteric switch that responds to the feedback inhibitor UDP-alpha-D-xylose
-
the structure of UGDH in the crystal form reveals a hexameric arrangement, composed a trimer of dimers of six subunits
-
sitting-drop vapour-diffusion method, diffraction quality crystals (maximum dimensions of 0.1 * 0.1 * 0.1 mm) are obtained within one week at 273C using reservoir solution composed of 4.0 M NaCl, 100 mM HEPES pH 7.5. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 117.7, b = 76.7, c = 75.6 A, beta = 125.8. Crystal structure is at a resolution of 2.0 A. The overall fold is comprised of an N-terminal NAD+ dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long alpha-helix
-
purified recombinant wild-type and selenomethionine-labeled UgdG at 4.5 and 5.5 mg/ml, respectively, in 25 mM Tris-HCl, pH 8.3, 50 mM NaCl, 2.5 mM DTT and 1 mM NAD+, or 0.5 mM UDP-GlcA and 1 mM NAD+, 0.0005 ml of each protein and precipitant solution are mixed at 20C, 24 h, the precipitant solution contains 200 mM Li2SO4, 100 mM Tris-HCl, pH 8.5, and 30% v/v PEG 4000, or 100 mM sodium citrate, pH 5.6, 20% 2-propanol, and 20% v/v PEG 4000, vapour diffusion method, method optimization, X-ray diffraction structure determination and analysis at 2.4 A resolution for the wild-type enzyme, and at 3.4 A resolution for the SeMet-UDG
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
-
80C, 10 min, enzyme retains full activity
721871
5.4 - 6.5
-
enzyme retains 93-97% of initial activity
725729
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
after incubation for 1.6 h loss of 40% activity
40
-
rapid and irreversible inactivation above
50
-
without substrate, complete inactivation
80
-
10 min, stable
90
-
half-life: 10 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
2-mercaptoethanol stabilizes
-
fairly stable with 2 mM dithiothreitol
-
NAD+ protects against heat inactivation
-
stable during repeated freezing and thawing cycles with 10% glycerol
-
UDPglucose protects against heat inactivation
-
UDPglucose stabilizes
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-10C, little loss of activity after several weeks
-
-12C, 2 weeks
-
-4C, frozen in presence of UDPglucose, stable for 2 months
-
-70C, 5 mM UDPglucose, 1 mM dithiothreitol
-
5C, 50 mM Tris-HCl, pH 8.7, 2 mM dithiothreitol, after 24 h 90% activity left
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel affinity and gel filtration; by nickel affinity and gel filtration; by nickel affinity and gel filtration
by nickel-NTA chromatography
-
partial
recombinant enzyme
recombinant glutathione-S-transferase fusion protein
-
recombinant His6-tagged BceC from Escherichia coli by affinity chromatography
-
recombinant His6-tagged UGDH from Escherichia coli by nickel affinity chromatography
-
recombinant His6-tagged wild-type and selenomethionine-labeled UgdG from Escherichia coli BL21 and B843, respectively by affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expression in Agrobacterium tumefaciens; expression in Agrobacterium tumefaciens; expression in Agrobacterium tumefaciens; expression in Agrobacterium tumefaciens
expression in Escherichia coli
expression in Escherichia coli; expression in Escherichia coli
expression in Pichia pastoris
expression of isoforms Ugd2, Ugd3, Ugd4 in Escherichia coli
-
expression of mutant enzyme wild-type enzymes in Escherichia coli
-
gene bceC, DNA and amino acid sequence determination and analysis, expression in Escherichia coli
-
gene Ugd(BCAL2946) cloned into the pGEM-T Easy vector, sequenced, and ligated into pET29a vector to transform Escherichia coli Top10 cells. Ugd(BCAL2946) expressed in Escherichia coli HMS 174(DE3); gene Ugd(BCAM0855) cloned into the pGEM-T Easy vector, sequenced, and ligated into pET29a vector to transform Escherichia coli Top10 cells. Ugd(BCAM0855) expressed in Escherichia coli HMS 174(DE3); gene Ugd(BCAM2034) cloned into the pGEM-T Easy vector, sequenced, and ligated into pET29a vector to transform Escherichia coli Top10 cells. Ugd(BCAM2034) expressed in Escherichia coli BL21 (DE3)
gene ugdG, expression of His6-tagged wild-type and selenomethionine-labeled UgdG in Escherichia coli BL21 and B843, respectively
genes szHasA (hyaluronan synthase gene) and szHasB (UDP-glucose-6-dehydrogenase gene) introduced into Lactococcus lactis strain NZ9000 under the control of nisA promoter and lacA promoter respectively, resulting in a dual-plasmid controlled expression system
-
hUGDH gene is cloned from a LNCaP cDNA library, from LNCaP C33 and C81 cells, expression of His6-tagged UGDH in Escherichia coli, expression of UGDH mutant D280N in HEK.293 cells
-
overexpression as glutathione-S-transferase fusion protein in Escherichia coli
-
overexpression in Escherichia coli JM109
-
overexpression of the enzyme from Xenopus laevis in human smooth muscle cells increases the accumulation of hyaluronan
recombinant UGDH expressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
dihydrotestosterone increases UGDH expression 2.5fold in androgen-dependent cells. However, upregulation of UGDH does not affect hyaluronan synthase expression or enhance hyaluronan production
-
expression of Ugd is induced by polymyxin B through RppA, a putative response regulator of the bacterial two-component system
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y10F
mutation in GXGYXG consensus motif, 9% residual activity. Tyr10 plays a catalytic role in the final hydrolysis step. Upon release of NADH after the second oxidation step, Tyr10 may work as a proton conveyer from the aqueous hydrogen-bonding proton wire system to the hydrolytic site
Y10K
mutation in GXGYXG consensus motif, 2% residual activity
Y10S
mutation in GXGYXG consensus motif, 3% residual activity
A222Q/S233G
C276E
-
activity is not measurable at pH 8.7, 22C
C276G
-
activity is not measurable at pH 8.7, 22C
C276K
-
activity is not measurable at pH 8.7, 22C
C276L
-
activity is not measurable at pH 8.7, 22C
C276Y
-
activity is not measurable at pH 8.7, 22C
D280A
-
extremely poor enzymic activity
D280E
-
site-directed mutagenesis, 3-fold increase in Km for UDP-glucose and a 2-fold reduced Vmax relative to that of the wild type
DELTA132
-
deletion of residue Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The crystal structure of the deletion construct reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. The open conformation stabilizes the construct as a hexamer with point group symmetry 32, similar to that of the active complex. The DELTA132 and UDP-alpha-D-xylose-inhibited structures have similar hexamer-building interfaces
E161Q
-
hydrolysis step becomes completely rate-limiting so that a thioester enzyme intermediate accumulates at steady state. Crystallization of E161Q in the presence of 5 mM UDP-glucose and 2 mM NAD results in trapping a thiohemiacetal enzyme intermediate
G13E
normal expression and stability of mutant, no enzymic activity, no photoaffinity labeling with nicotinamide 2-azidoadenosine dinucleotide
K220H
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site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function
K220R
-
site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function
N224A
-
steady-state kinetic parameters are within an order of magnitude of the native enzyme
T131S
-
steady-state kinetic parameters are within an order of magnitude of the native enzyme
C260A
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no oxidation of UDP-glucose to glucuronic acid, but capable of both reducing the aldehyde intermediate and oxidizing the hydrated form of the aldehyde intermediate, protein is expressed in inclusion bodies
E141Q
-
kcat-value 10fold lower than wild-type
E145Q
-
kcat-value 10fold lower than wild-type
T118A
-
160fold reduction of kcat value
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
changes in the mRNA level during peach fruit development correspond to changes in the amount of cell wall material and the cell wall uronic acid content. These are greater in the fruits of the commercial cultivars compared with the Japanese native peach cultivars, and the expression of enzyme is higher in the fruits of the commercial cultivars
medicine
pharmacology
-
target for inhibitor design
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