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Information on EC 5.1.3.2 - UDP-glucose 4-epimerase and Organism(s) Escherichia coli and UniProt Accession P09147
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5.1.3.2 UDP-glucose 4-epimeraseIUBMB Comments
Requires NAD+. Also acts on UDP-2-deoxyglucose.
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Word Map
- 5.1.3.2
- polysaccharide
- galactokinase
-
leloir
- galactosemia
- galactose-1-phosphate
- udp-galnac
- udp-n-acetylglucosamine
-
galactose-inducible
-
kluyveromyces
-
pyrophosphorylase
- fragilis
-
uridylyltransferase
- udp-n-acetylgalactosamine
- udpglucose
- udp-sugars
- galnac
- mutarotase
-
galactose-containing
-
o-antigens
-
udp-glucuronic
- medicine
- synthesis
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
gal10, udp-galactose 4-epimerase, 4-epimerase, udp-glucose 4-epimerase, udp-galactose-4-epimerase, udp-galactose 4'-epimerase, udp-glucose 4'-epimerase, udp-glucose-4-epimerase, udp-glucose epimerase, gal10p, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
4-Epimerase
-
-
-
-
Epimerase, uridine diphosphoglucose
-
-
-
-
Galactowaldenase
-
-
-
-
UDP-D-galactose 4-epimerase
-
-
-
-
UDP-galactose 4-epimerase
UDP-glucose epimerase
-
-
-
-
UDPG-4-epimerase
-
-
-
-
UDPgalactose 4-epimerase
-
-
-
-
Uridine diphosphate galactose 4-epimerase
-
-
-
-
Uridine diphosphate glucose 4-epimerase
-
-
-
-
Uridine diphospho-galactose-4-epimerase
-
-
-
-
Uridine diphosphoglucose 4-epimerase
-
-
-
-
Uridine diphosphoglucose epimerase
-
-
-
-
UDP-galactose 4-epimerase
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-glucose = UDP-alpha-D-galactose
revolving door reaction mechanism, Tyr149 is the base catalyst for hydride transfer, overview. The enzyme undergoes a conformational change upon binding of the UDP sugar, which is in fact a result of the binding of the UMP-moiety of the substrate. The conserved lysine from the YxxxK motif plays an important role in the activation of the cofactor, as due to the conformational change, the 6-ammonium group is hydrogen-bonded to both the 2'- and 3'-hydroxylgroups of the nicotinamide riboside of NAD+
UDP-alpha-D-glucose = UDP-alpha-D-galactose
a nucleoside diphospho-4-ulose of the configuration D-xylo-4-hexosulose is an intermediate in the reaction
-
UDP-alpha-D-glucose = UDP-alpha-D-galactose
structurereactivity studies and reaction mode, acid-base catalysis of hydride transfer, overview
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
catalyses the interconversion of UDP-Gal and UDPGlc
PATHWAY SOURCE
PATHWAYS
BRENDA
KEGG
-
-, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
UDP-glucose 4-epimerase
Requires NAD+. Also acts on UDP-2-deoxyglucose.
CAS REGISTRY NUMBER
COMMENTARY
9032-89-7
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
D-galactose
D-glucose
the catalytic efficiencies of GalE for D-galactose is much lower than for UDP-galactose
-
-
?
D-tagatose
D-fructose
the catalytic efficiencies of GalE for D-tagatose is much lower than for UDP-galactose
-
-
?
UDP-alpha-D-glucose
UDP-alpha-D-galactose
-
with NAD+ as cofactor, UDP-4-ketopyranose and NADH are reaction intermediates. Weak binding of the 4-ketopyranosyl moiety and strong binding of the UDP-moiety within a substrate domain allow either face of the 4-ketopyranosyl moiety to accept hydride from NADH, pH-dependent charge transfer complex between Tyr149 and NAD+
-
-
?
UDP-galactose
UDP-glucose
-
role of the essential arginine residue in stretching and binding of the substrate
-
?
UDP-galactose
UDP-glucose
-
enzyme catalyzes the first step of the Lelpoir pathway
-
-
r
UDP-GlcNAc
UDP-GalNAc
-
wild-type enzyme shows no interconversion of UDP-GlcNAc and UDP-GalNAc, mutation Y299C results in a gain of activity against UDP-GalNAc by more than 230fold
-
r
additional information
?
-
no 4-epimerization activity is found for allose, gulose, altrose, idose, mannose, and talose
-
-
?
additional information
?
-
-
no 4-epimerization activity is found for allose, gulose, altrose, idose, mannose, and talose
-
-
?
additional information
?
-
Escherichia coli GalE is unable to catalyse the epimerization of acetylated substrates due to the so-called gatekeeper wall of the active site substrate-binding hexagonal box that is occupied by a bulky residue, Tyr299
-
-
?
additional information
?
-
-
TDP-[4-3H]glucose donates tritium to enzyme-bound NAD+, leading to formation of enzyme-[3H1]NADH. This suggests that a nucleoside diphospho-4-ulose of the configuration D-xylo-4-hexosulose is an intermediate in the reaction
-
-
?
additional information
?
-
-
no interconversion of UDP-GalNAc and UDP-GlcNAc
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
UDP-galactose
UDP-glucose
-
enzyme catalyzes the first step of the Lelpoir pathway
-
-
r
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
coenzyme is tightly bound at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys153 and nicotinamide N1 of NAD+
NAD+
two paired Rossmann folds tightly bind one NAD+ cofactor per subunit. In Escherichia coli GalE, the NAD+ interacts more extensively with the protein than is observed with other SDR enzymes. pH-Dependent charge transfer complex between Tyr149 and NAD+
NAD+
-
two nicotinamide cofactors bind in symmetry-related positions in the dimer
NAD+
-
NAD+ is very tightly but noncovalently bound in the active site, NAD+ is reduced to NADH in the course of catalysis. NADH associated with the purified enzyme is a component of the inactive, abortive complexes, enzyme-NADH-uridine nucleotide, that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth
NAD+
-
the fully active GalE is dimeric and contains one tightly bound NAD+ per subunit, NAD+ undergoes reversible reduction to NADH in the chemical mechanism, practically irreversible binding of NAD+ within a Rossmann-type fold, nonstereospecific hydride transfer, uridine nucleotide-induced activation of NAD, Tyr149 as a base catalyst, and [GalE-NADH]-oxidation in one-electron steps by one-electron acceptors. pH-Dependent charge transfer complex between Tyr149 and NAD+. Binding structure, overview
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5'-UMP
-
the native enzyme is completely insensitive to inhibition, the desensitized enzyme is strongly inhibited. Desensitization by heat converts the enzyme to its ultimate catalytic form
L-Arabinose plus UMP or UDP
-
NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP
-
Sodium cyanoborohydride
-
NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction, mutant proteins K153M and K153A bind UMP very well, but the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP
UDP-6-deoxygalactose
-
weak competitive inhibitor with respect to UDPgalactose
uridine-5'-diphosphoro-beta-1-(5-sulfonic acid)naphthylamidate
-
powerful competitive
glucose plus UMP
-
NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP
-
NADH
-
NADH associated with the purified enzyme is a component of the inactive, abortive complexes, enzyme-NADH-uridine nucleotide, that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth
additional information
-
no inactivation by the UDP-D-fucose or D-fucose alone or by UDP-D-fucose plus 5'-UMP
-
additional information
-
NAD+ associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride. The mutant protein binds UMP very well, but the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP. The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site, however the purified mutants K153M and K153A contain very little NADH
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2 - 6
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant Y149F
83
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant K153M
110
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant S124A
230
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant wild-type enzyme
260
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant S124T
0.14
UDPgalactose
-
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.073
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant Y149F
0.61
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant S124A
0.67
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant K153M
250
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant S124T
760
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant wild-type enzyme
960
UDPgalactose
-
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2.9
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant Y149F
5.5
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant S124A
8.1
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant mutant K153M
970
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant S124T
3400
UDP-alpha-D-glucose
-
pH 6.2, temperature not specified in the publication, recombinant wild-type enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5 - 8.3
-
activity of the wild-type enzyme is pH-independent in the pH-range of 5.5-9.3
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
UDP-Gal 4-epimerases and the other GalE-like UDP-sugar 4-epimerases belong to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Classification of UDP-hexose 4-epimerases into three groups with distinct substrate promiscuity. Group 1 contains the 4-epimerases that exhibit a strong preference for non-acetylated substrates (such as Escherichia coli enzyme eGalE), group 2 members can epimerize both non-acetylated and N-acetylated substrates equally well (such as the human enzyme hGalE), and group 3 epimerases are very specific for N-acetylated substrates (like the WbpP from Pseudomonas aeruginosa). Despite the relatively low sequence identity among all three groups, the similarity of the enzymes' tertiary structures is striking with an overall RMSD of the multiple structure alignment being 1.08 A and variation being most pronounced at the C-terminal end
malfunction
the S306Y mutation allows a switch from group 2 to group 1 and forms steric clashes between the group 3 epimerases and their substrates, which results in the observed loss of activity
metabolism
UDP-sugar 4-epimerase (GalE) is one of enzymes in the Leloir pathway
physiological function
UDP-galactose 4-epimerase is important for the biosynthesis of polysaccharide structures, such as capsular polysaccharide (CPS)
physiological function
-
UDP-Gal provides all galactosyl units in biologically synthesized carbohydrates. All healthy cells produce UDP-Gal from uridine 5'-diphospho-alpha-D-glucose UDP-Glc, by the action of UDP-galactose 4-epimerase
additional information
additional information
enzyme structure and substrate specificity, structure-function relationship, overview. Comparison of the hexagonal box model of sugar-binding pockets of several GalE enzymes
additional information
-
ligand-bound enzyme structures, active site structure including Lys153, Tyr149, and Ser124, overview
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
160000
-
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker, dimeric form, gel filtration
320000
-
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker, tetrameric form, gel filtration
39000
40000
79000
80000
39000
-
1 * 39000, subunits of the dimeric epimerase are stabilized under certain conditions where they can function catalytically
80000
-
2 * 80000, fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker exist as monomeric, dimeric and tetrameric form. The monomeric form has low epimerase activity, SDS-PAGE
80000
-
4 * 80000, fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker exist as monomeric, dimeric and tetrameric form. The monomeric form has low epimerase activity, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
monomer
tetramer
additional information
the enzyme has an N-terminal nucleotide binding domain and a smaller C-terminal domain that is responsible for the correct positioning of its substrate, a UDP-sugar. The N-terminal domain comprises seven parallel beta-strands that are flanked on both sides by alpha-helices and shape the Rossmann fold. Two paired Rossmann folds tightly bind one NAD+ cofactor per subunit
dimer
-
2 * 80000, fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker exist as monomeric, dimeric and tetrameric form. The monomeric form has low epimerase activity, SDS-PAGE
monomer
-
2 * 80000, fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker exist as monomeric, dimeric and tetrameric form. The monomeric form has low epimerase activity, SDS-PAGE
monomer
-
1 * 39000, subunits of the dimeric epimerase are stabilized under certain conditions where they can function catalytically
tetramer
-
4 * 80000, fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker exist as monomeric, dimeric and tetrameric form. The monomeric form has low epimerase activity, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structure of the oxidized and reduced forms of UDP-galactose 4-epimerase
crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K153M
mutation results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild-type and 1.8 ppm upfield from that of Y149F epimerase
N179S
the 4-epimerization of tagatose is enhanced 2fold in this mutant
S124A
S124A/Y149F
mutation causes a 13C downfield perturbation of 2.8 ppm to 152.7 ppm
S124T
decrease in activity of the mutant enzymes S124A, S124T, and S124V is due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural pertubations
S124V
decrease in activity of the mutant enzymes S124A, S124T, and S124V is due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural pertubations
S143A
site-directed mutagenesis, the mutation abolishes activity on non-acetylated substrates, probably due to loss of the hydrogen bonding, whereas the mutant remains active on UDP-GlcNAc/UDP-GalNAc, as additional stabilizing interactions with the N-acetyl moiety are present
S306Y
site-directed mutagenesis, the mutation allows a switch from group 2 to group 1 and forms steric clashes between the group 3 epimerases and their substrates,which results in the observed loss of activity
Y149F
mutation results in a 13C downfield perturbation of 2.7 ppm to 152.6 ppm
Y299C
site-directed mutagenesis, structure analysis in complex with UDP-N-acetylglucosamine, PDB ID 1LRK, the Y299C mutation in eGalE results in significant loss of activity on non-acetylated substrates
K153A
-
NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP. NAD+ associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride. The mutant protein binds UMP very well, but the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP. The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site, however the purified mutants K153M and K153A contain very little NADH
K153M
S124A
S124A/Y149F
-
epimerization proceeds at a turnover number that is lower by a factor of 10000000 than that of the wild-type enzyme. This is attributed to the synergistic action of Tyr149 and Ser124 in wild-type enzyme and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme. 80% inactivation after 8 min at 50°C compared to 20% inactivation of the wild-type enzyme
S124T
S124V
-
mutant forms Y149F, S124A, S124V, and S124T. The least active mutant is Y149F, with a turnover number 0.010% of that for the wild type enzyme. The activity of S124A is also very low, with a turnover number 0.035% of that of the wild type enzyme. The Km values of Y149F and S124A are 12% and 21% of that of the wild type enzyme, respectively. The turnover number for S124T is about 30% of that of the wild type enzyme, and the Km value is similar. Second-order rate constants for reductive inactivation by NaBH3CN are similar to that for the wild type enzyme in the cases of S124A, S124T, and S124V. Y149F reacts with NaBH3- 12-20fold faster than the wild type enzyme at pH 8.5 and 7.0, respectively
S306Y
-
plasmid containing the Gne S306Y constructed using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA).The S306Y mutation totally abolished activity toward the acetylated substrate.
Y149F
Y299C
-
mutation results in a loss of epimerase activity with regard to UDPgalactose by almost 5fold, it results in a gain of activity against UDP-GalNAc by more than 230fold
additional information
-
second-order rate constants for reductive inactivation of wild-type and mutant epimerases, overview
S124A
decrease in activity of the mutant enzymes S124A, S124T, and S124V is due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural pertubations
K153M
-
NAD+ associated with the wild type enzyme is subject to UMP-dependent reduction by sugars such as glucose and arabinose, but the mutant proteins K153M and K153A are not reduced by sugars in the presence or absence of UMP. NAD+ associated with the wild type enzyme is also subject to UMP-dependent reduction by sodium cyanoborohydride. The mutant protein binds UMP very well, but the rate at which NAD+ associated with them is reduced by sodium cyanoborohydride is almost insensitive to the presence of UMP. The purified wild type enzyme contains significant amounts of NADH bound to the coenzyme site, however the purified mutants K153M and K153A contain very little NADH
K153M
-
site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
S124A
-
mutant forms Y149F, S124A, S124V, and S124T. The least active mutant is Y149F, with a turnover number 0.010% of that for the wild type enzyme. The activity of S124A is also very low, with a turnover number 0.035% of that of the wild type enzyme. The Km values of Y149F and S124A are 12% and 21% of that of the wild type enzyme, respectively. The turnover number for S124T is about 30% of that of the wild type enzyme, and the Km value is similar. Second-order rate constants for reductive inactivation by NaBH3CN are similar to that for the wild type enzyme in the cases of S124A, S124T, and S124V. Y149F reacts with NaBH3- 12-20fold faster than the wild type enzyme at pH 8.5 and 7.0, respectively
S124A
-
in contrast to wild-type enzyme the mutant enzyme displays a significant deuterium kinetic isotope effect. Epimerization proceeds with a deuterium kinetic isotope effect of about 2 throughout the pH range 6.3-9.0
S124A
-
site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
S124T
-
mutant forms Y149F, S124A, S124V, and S124T. The least active mutant is Y149F, with a turnover number 0.010% of that for the wild type enzyme. The activity of S124A is also very low, with a turnover number 0.035% of that of the wild type enzyme. The Km values of Y149F and S124A are 12% and 21% of that of the wild type enzyme, respectively. The turnover number for S124T is about 30% of that of the wild type enzyme, and the Km value is similar. Second-order rate constants for reductive inactivation by NaBH3CN are similar to that for the wild type enzyme in the cases of S124A, S124T, and S124V. Y149F reacts with NaBH3- 12-20fold faster than the wild type enzyme at pH 8.5 and 7.0, respectively
S124T
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y149F
-
mutant forms Y149F, S124A, S124V, and S124T. The least active mutant is Y149F, with a turnover number 0.010% of that for the wild type enzyme. The activity of S124A is also very low, with a turnover number 0.035% of that of the wild type enzyme. The Km values form Y149F and S124A are 12% and 21% of that of the wild type enzyme, respectively. The turnover number for S124T is about 30% of that of the wild type enzyme, and the Km value is similar. Second-order rate constants for reductive inactivation by NaBH3CN are similar to that for the wild type enzyme in the cases of S124A, S124T, and S124V. Y149F reacts with NaBH3- 12-20fold faster than the wild type enzyme at pH 8.5 and 7.0, respectively
Y149F
-
in contrast to wild-type enzyme the mutant enzyme displays a significant deuterium kinetic isotope effect. At pH there is no significant isotope effect, but at pH 6.3, the isotope effect is 2.2
Y149F
-
site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
50
50
-
wild-type enzyme loses 20% of its activity after 8 min, mutant enzyme S124A/Y149F loses 80% of its activity after 8 min
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
urea
-
denaturation by 8 M urea at pH 7.0 causes 85% loss of ist secondary structure and dissociation of its constituent molecules
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in GALE-null line of Chinesese hamster ovary cells. Enzyme from Escherichia coli can not restore the metabolic balance, because unlike the mammalian enzyme it can not catalyze the interconversion of UDP-GalNAc and UDP-GlcNAc
-
fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
denatured by 8 M urea at pH 7.0 to a state having 15% of residual secondary structure. Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme. Reactivation follows a socond-order kinetics
-
dilution of the denaturant urea by buffer at pH 8.5 leads to functional reconstitution of the dimeric holoenzyme. The refolding process is biphasic: after 2 min an equilibrium conformer is formed having 72% of its native secondary structure and by 60 min reactivation becomes complete. The early intermediate has lower energy of activation against thermal denaturation than the reactivated state
-
purified enzyme dimer consists of a mixture of catalytically active subunits designated enzyme-NAD+ and inactive, abortive complexes designated enzyme-NADH-uridine nucleotide, in which the uridine nucleotide may be UDPglucose, UDPgalactose, or UDP. The abortive complexes are transformed into active enzyme-NAD+ by denaturation of the purified enzyme at 4°C in 6 M guanidine hydrochloride buffered at pH 7.0 in the presence of 0.126 mM NAD+ for 3 h, followed by dilution of guanidine hydrochloride to 0.18 M and of NAD+ to 0.076 mM for 2 h. The renatured enzyme is fully active and contains negligible amounts of NADH and uridine nucleotides
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
preparation of a fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-phosphate uridylyltransferase with an intervening Ala3 linker, shows kinetic advantages in that the initial velocity to produce glucose 1-phosphate from UDPgalactose an
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Arabshahi, A.; Flentke, G.R.; Frey, P.A.
Uridine diphosphate galactose 4-epimerase. pH-Dependence of the reduction of NAD+ by a substrate analog
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1988
Escherichia coli
Blackburn, P.; Ferdinand, W.
The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar
Biochem. J.
155
225-229
1976
Escherichia coli
Gabrial, O.; Kalckar, H.M.; Darrow, R.A.
UDP-galactose-4-epimerase
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(Ebner, K. B., ed.)
2
85-135
1975
Bos taurus, Kluyveromyces marxianus, Escherichia coli, Vigna radiata var. radiata, Saccharomyces fragilis, Torulopsis candida
-
Adair, W.L.; Gabriel, O.; Ullrey, D.; Kalckar, H.M.
4-Uloses as intermediates in enzyme-nicotinamide adenine dinucleotide-mediated oxidoreductase mechanisms
J. Biol. Chem.
248
4635-4639
1973
Escherichia coli
Spencer, M.; Blackburn, P.; Ferdinand, W.; Blackburn, G.M.
Competitive inhibition and substrate activity of uridine diphosphate 6-deoxygalactose for Escherichia coli uridine diphosphate galactose 4-epimerase
Biochem. J.
131
421-423
1973
Escherichia coli
Wilson, D.B.; Hogness, D.S.
The enzymes of the galactose operon in Escherichia coli. I. Purification and characterization of uridine diphosphogalactose 4-epimerase
J. Biol. Chem.
239
2469-2481
1964
Escherichia coli
Vorgias, C.E.; Lemaire, H.G.; Wilson, K.S.
Overexpression and purification of the galactose operon enzymes from Escherichia coli
Protein Expr. Purif.
2
330-338
1991
Escherichia coli
Bauer, A.J.; Rayment, I.; Frey, P.A.; Holden, H.M.
The isolation, purification, and preliminary crystallographic characterization of UDP-galactose-4-epimerase from Escherichia coli
Proteins Struct. Funct. Genet.
9
135-142
1991
Escherichia coli
Vanhooke, J.L.; Frey, P.A.
Characterization and activation of naturally occuring abortive complexes of UDP-galactose 4-epimerase from Escherichia coli
J. Biol. Chem.
269
31596-31504
1994
Escherichia coli
-
Tamada, Y.; Swanson, B.A.; Arabshahi, A.; Frey, P.A.
Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase
Bioconjug. Chem.
5
660-665
1994
Escherichia coli
Liu, Y.; Vanhooke, J.L.; Frey, P.A.
UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition
Biochemistry
35
7615-7620
1996
Escherichia coli
Liu, Y.; Thoden, J.B.; Kim, J.; Berger, E.; Gulick, A.M.; Ruzicka, F.J.; Holden, H.M.; Frey, P.A.
Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli
Biochemistry
36
10675-10684
1997
Escherichia coli
Nayar, S.; Bhattacharyya, D.
UDP-galactose 4-epimerase from Escherichia coli: existence of a catalytic monomer
FEBS Lett.
409
449-451
1997
Escherichia coli
Dutta, S.; Maiti, N.R.; Bhattacharyya, D.
Reversible folding of UDP-galactose 4-epimerase from Escherichia coli
Eur. J. Biochem.
244
407-413
1997
Escherichia coli
Flentke, G.R.; Frey, P.A.
Reaction of uridine diphosphate galactose 4-epimerase with a suicide inactivator
Biochemistry
29
2430-2436
1990
Escherichia coli
Thoden, J.B.; Gulick, A.M.; Holden, H.M.
Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli
Biochemistry
36
10685-10695
1997
Escherichia coli (P09147), Escherichia coli
Thoden, J.B.; Frey, P.A.; Holden, H.M.
High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol
Protein Sci.
5
2149-2161
1996
Escherichia coli
Thoden, J.B.; Frey, P.A.; Holden, H.M.
Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism
Biochemistry
35
5137-5144
1996
Escherichia coli (P09147), Escherichia coli
Thoden, J.B.; Frey, P.A.; Holden, H.M.
Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli
Biochemistry
35
2557-2566
1996
Escherichia coli (P09147), Escherichia coli
Swanson, B.A.; Frey, P.A.
Identification of lysine 153 as a functionally important residue in UDP-galactose 4-epimerase from Escherichia coli
Biochemistry
32
13231-13236
1993
Escherichia coli
Bauer, A.J.; Rayment, I.; Frey, P.A.; Holden, H.M.
The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution
Proteins
12
372-381
1992
Escherichia coli
Barat, B.; Bhattacharyya, D.
UDP-galactose 4-epimerase from Escherichia coli: formation of catalytic site during reversible folding
Arch. Biochem. Biophys.
391
188-196
2001
Escherichia coli
Wei, Y.; Lin, J.; Frey, P.A.
13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides
Biochemistry
40
11279-11287
2001
Escherichia coli (P09147)
Berger, E.; Arabshahi, A.; Wei, Y.; Schilling, J.F.; Frey, P.A.
Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149
Biochemistry
40
6699-6705
2001
Escherichia coli
Liu, Y.; Arabshahi, A.; Frey, P.A.
Rate enhancements brought about by uridine nucleotides in the reduction of NAD+ at the active site of UDP-galactose 4-epimerase
Bioorg. Chem.
28
29-37
2000
Escherichia coli
-
Chen, X.; Kowal, P.; Hamad, S.; Fan, H.; Wang, P.G.
Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli
Biotechnol. Lett.
21
1131-1135
1999
Escherichia coli
-
Bhattacharyya, U.; Dhar, G.; Bhaduri, A.
An arginine residue is essential for stretching and binding of the substrate on UDP-glucose-4-epimerase from Escherichia coli. Use of a stacked and quenched uridine nucleotide fluorophore as probe
J. Biol. Chem.
274
14573-14578
1999
Escherichia coli
Holden, H.M.; Rayment, I.; Thoden, J.B.
Structure and function of enzymes of the Leloir pathway for galactose metabolism
J. Biol. Chem.
278
43885-43888
2003
Escherichia coli, Homo sapiens
Schulz, J.M.; Ross, K.L.; Malmstrom, K.; Krieger, M.; Fridovich-Keil, J.L.
Mediators of galactose sensitivity in UDP-galactose 4'-epimerase-impaired mammalian cells
J. Biol. Chem.
280
13493-13502
2005
Escherichia coli, Homo sapiens
Guo, H.; Yi, W.; Li, L.; Wang, P.G.
Three UDP-hexose 4-epimerases with overlapping substrate specificity coexist in E. coli O86:B7
Biochem. Biophys. Res. Commun.
356
604-609
2007
Escherichia coli
Guo, H.; Li, L.; Wang, P.G.
Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7
Biochemistry
45
13760-13768
2006
Escherichia coli, Escherichia coli O86:B7
Zhang, H.C.; Bi, J.Y.; Chen, C.; Huang, G.L.; Qi, Q.S.; Xiao, M.; Wang, P.G.
Immobilization of UDP-galactose 4-epimerase from Escherichia coli on the yeast cell surface
Biosci. Biotechnol. Biochem.
70
2303-2306
2006
Escherichia coli
Kim, H.J.; Kang, S.Y.; Park, J.J.; Kim, P.
Novel activity of UDP-galactose-4-epimerase for free monosaccharide and activity improvement by active site-saturation mutagenesis
Appl. Biochem. Biotechnol.
163
444-451
2011
Escherichia coli (P09147), Escherichia coli
Frey, P.A.; Hegeman, A.D.
Chemical and stereochemical actions of UDP-galactose 4-epimerase
Acc. Chem. Res.
46
1417-1426
2013
Escherichia coli
Beerens, K.; Soetaert, W.; Desmet, T.
UDP-hexose 4-epimerases a view on structure, mechanism and substrate specificity
Carbohydr. Res.
414
8-14
2015
Marinithermus hydrothermalis (F2NQX6), Thermus thermophilus (F6DEY6), Saccharomyces cerevisiae (P04397), Escherichia coli (P09147), Streptococcus thermophilus (P21977), Homo sapiens (Q14376), Homo sapiens, Drosophila melanogaster (Q9W0P5), Saccharomyces cerevisiae ATCC 204508 / S288c (P04397), Thermus thermophilus SG0.5JP17-16 (F6DEY6), Marinithermus hydrothermalis DSM 14884 / JCM 11576 / T1 (F2NQX6)
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