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M134A
site-directed mutagenesis
M134Y
site-directed mutagenesis
M134A
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site-directed mutagenesis
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M134Y
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site-directed mutagenesis
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K153N
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the mutation affects the catalytic activity only slightly, however, the NAD+ binding potential is reduced dramatically
Y149G
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the mutant is completely inactive
Y149G/K153N
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the mutant is completely inactive
K160V
site-directed mutagenesis of the key residue responsible for anchoring of the co-factor, inactive mutant
L320C
site-directed mutagenesis of a gate-keeper residue, the L320C mutant enzyme is also active in UDPGlcpNAc/UDP-GalpNAc interconversion
L320Y
site-directed mutagenesis of a gate-keeper residue, the L320Y mutant enzyme is not active in UDPGlcpNAc/UDP-GalpNAc interconversion
Y156F
site-directed mutagenesis of the key residue serving as the active site base, inactive mutant
K153A
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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
N179S
the 4-epimerization of tagatose is enhanced 2fold in this mutant
S124A/Y229F
site-directed mutagenesis, inactive mutant
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
S144K
site-directed mutagenesis, inactive mutant
S306Y
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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.
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A25V
the mutant shows reduced activity compared to the wild-type enzyme
C307Y
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normal activity with respect to UDP-galactose, complete loss of activity with respect to UDP-N-acetylgalactosamine
D69E
the mutant shows reduced activity compared to the wild-type enzyme
G302D
the mutant enzyme is not able to rescue galactose-sensitive cell proliferation when stably expressed in ldlD cells
G319E
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very littel change in steady-state kinetic parameters compared with the wild-type protein
K257R
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the ratio of turnover number to Km-value is 6.7fold lower than the wild-type ratio
L313M
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the ratio of turnover number to Km-value is 3.0fold lower than the wild-type ratio
M284K
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the mutant is active in vivo, but not in vitro and shows reduced enzymatic activity (1.1% residual activity) and reduced stability towards denaturants in vitro
N268D
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the mutant demonstrates 63% residual activity
R169W
the mutant shows reduced activity compared to the wild-type enzyme
R239W
the mutant enzyme is not able to rescue galactose-sensitive cell proliferation when stably expressed in ldlD cells
R40C
the mutant shows reduced activity compared to the wild-type enzyme
S132A
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complete loss of activity with respect to interconversion of UDP-glucose and UDP-galactose and of UDP-GalNAc and UDP-GlcNAc
S132A/Y157F
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complete loss of activity with respect to interconversion of UDP-glucose and UDP-galactose and of UDP-GalNAc and UDP-GlcNAc
Y105C
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the mutant demonstrates 13% residual activity
Y157F
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complete loss of activity with respect to interconversion of UDP-glucose and UDP-galactose and of UDP-GalNAc and UDP-GlcNAc
G118A/G119A
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site-directed mutagenesis, the mutant shows highly reduced activity with UDP-N-acetylglucosamine and reduced activity with UDP-Gal, the mutant's substrate specificity is shifted toward non-acetylated substrates
G188S/G119S
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site-directed mutagenesis, the mutant shows highly reduced activity with UDP-N-acetylglucosamine and reduced activity with UDP-Gal, the mutant's substrate specificity is shifted toward non-acetylated substrates
S116A
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site-directed mutagenesis, the mutant shows highly reduced activity with UDP-N-acetylglucosamine and reduced activity with UDP-Gal, the mutant's substrate specificity is shifted toward non-acetylated substrates
S279Y
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site-directed mutagenesis, the mutant shows highly reduced activity with UDP-N-acetylglucosamine and reduced activity with UDP-Gal, the mutant's substrate specificity is shifted toward non-acetylated substrates
T117S
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site-directed mutagenesis, the mutant shows highly reduced activity with UDP-N-acetylglucosamine and reduced activity with UDP-Gal, the mutant's substrate specificity is shifted toward non-acetylated substrates
K150R
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
S121A
site-directed mutagenesis, inactive mutant
Y146F
site-directed mutagenesis, inactive mutant
K150R
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type
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S121A
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site-directed mutagenesis, inactive mutant
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Y146F
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site-directed mutagenesis, inactive mutant
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C300Y
site-directed mutagenesis, the mutation results in decreased activity toward UDP-GlcNAc and UDP-GalNAc
K86G
site-directed mutagenesis, the mutation abolishes the ability of the enzyme to transform UDP-Glc/UDP-Gal completely
C300Y
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site-directed mutagenesis, the mutation results in decreased activity toward UDP-GlcNAc and UDP-GalNAc
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K86G
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site-directed mutagenesis, the mutation abolishes the ability of the enzyme to transform UDP-Glc/UDP-Gal completely
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K151A
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site-directed mutagenesis, inactive mutant
S123A
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site-directed mutagenesis, inactive mutant
Y147A
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site-directed mutagenesis, almost inactive mutant
K151A
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site-directed mutagenesis, inactive mutant
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S123A
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site-directed mutagenesis, inactive mutant
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Y147A
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site-directed mutagenesis, almost inactive mutant
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N191D
naturally occuring mutation, the UgeA mutant shows reduced galactofuranose production, the mutant enzyme is partially active
N191D
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naturally occuring mutation, the UgeA mutant shows reduced galactofuranose production, the mutant enzyme is partially active
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K153M
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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
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
K153M
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site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
S124A
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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
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
S124A
site-directed mutagenesis
S124A
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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
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site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
S124A/Y149F
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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
S124A/Y149F
mutation causes a 13C downfield perturbation of 2.8 ppm to 152.7 ppm
S124T
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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
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
S124T
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
S124V
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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
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
S306Y
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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.
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
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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
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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
mutation results in a 13C downfield perturbation of 2.7 ppm to 152.6 ppm
Y149F
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site-directed mutagenesis, the mutant shows reduced highly activity compared to the wild-type enzyme
Y299C
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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
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
D103G
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the ratio of turnover number to Km-value is 14.4fold lower than the wild-type ratio
D103G
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the mutant demonstrates 82.1% residual activity
G90E
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the ratio of turnover number to Km-value is 1040fold lower than the wild-type ratio. Mutant enzyme is more susceptible to proteolysis than the wild-type protein, presence of substrate at saturating level (1 mM) partially protect the enzyme from proteolysis
G90E
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the mutant demonstrates 1% residual activity
L183P
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the ratio of turnover number to Km-value is 4.7fold lower than the wild-type ratio. Mutant enzyme is highly susceptible to proteolysis during expression and purification
L183P
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the mutant demonstrates 3.3% residual activity
N34S
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very little change in steady-state kinetic parameters compared with the wild-type protein. Mutant enzyme is more susceptible to proteolysis than the wild-type protein, presence of substrate at saturating level (1 mM) partially protect the enzyme from proteolysis
N34S
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the mutant demonstrates above 65.1% residual activity
R335H
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the ratio of turnover number to KM-value is 3.5fold lower than the wild-type ratio
R335H
the mutant shows reduced activity compared to the wild-type enzyme
V94M
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the ratio of turnover number to Km-value is 75fold lower than the wild-type ratio
V94M
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the mutant demonstrates 2.6% residual activity
additional information
identification of a mutant allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which is a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides, phenotype, overview
additional information
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identification of a mutant allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which is a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides, phenotype, overview
additional information
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identification of a mutant allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which is a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides, phenotype, overview
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additional information
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generation of single disruption mutants and DELTAuge3 DELTAuge5 double mutant, phenotypes, overview
additional information
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generation of single disruption mutants and DELTAuge3 DELTAuge5 double mutant, phenotypes, overview
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additional information
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a ugeADELTA knockout strain is viable, but has defects including wide, slow growing, highly branched hyphae and reduced conidiation. ugeADELTA colonies have substantially reduced sporulation but normal spore viability. Conidia of the ugeADELTA strain can not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings, suggesting they ceased to grow from starvation
additional information
targeted deletion of ugeA resulting in a severe reduction of galactofuranose in N-linked glucans
additional information
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targeted deletion of ugeA resulting in a severe reduction of galactofuranose in N-linked glucans
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additional information
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second-order rate constants for reductive inactivation of wild-type and mutant epimerases, overview
additional information
silencing gene GALE with specific siRNAs in human chondrocytes
additional information
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silencing gene GALE with specific siRNAs in human chondrocytes
additional information
addition of NAD+ to Lys150 significantly abrogates the loss of activity. None of the mutations affected the quaternary structure of the protein
additional information
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addition of NAD+ to Lys150 significantly abrogates the loss of activity. None of the mutations affected the quaternary structure of the protein
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additional information
development of transgenic rice plants to constitutively overexpress the OsUGE-1 gene (OsUGE1-OX1-2). The transgenic rice lines are similar in size to wild-type plants at the vegetative stage and at maturity regardless of the N-level tested. OsUGE1-OX lines maintain 18-24% more sucrose and 12-22% less cellulose in shoots compared to wild-type when subjected to suboptimal N-levels. OsUGE1-OX lines maintain proportionally more galactose and glucose in the hemicellulosic polysaccharide profile of plants compared to wild-type plants when grown under low N. The altered cell wall C-partitioning during N-limitation in the OsUGE1-OX lines appears to be mediated by OsUGE1 via the repression of the cellulose synthesis associated genes, OsSus1, OsCesA4, 7, and 9. Seed characteristics of rice plants overexpressing OsUGE1 compared to wild-type rice, overview
additional information
a GalE loop deletion mutant (mut1), in which residues 32-43 (NLSSGRREFVNP) of the NAD-binding loop are replaced with residues 33-40 (IVQRDTGG) of the corresponding loop from Thermoplasma volcanium binds NAD+ in a loose, reversible manner
additional information
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a GalE loop deletion mutant (mut1), in which residues 32-43 (NLSSGRREFVNP) of the NAD-binding loop are replaced with residues 33-40 (IVQRDTGG) of the corresponding loop from Thermoplasma volcanium binds NAD+ in a loose, reversible manner
additional information
construction of disruption mutants, DELTAuge1 and DELTAuge1DELTAgal10. Both mutant strains are sensitive to hygromycin B. Oligosaccharide content is reduced in acid phosphatase prepared from the uge1DELTA strain. The uge1DELTA strain grown in 0.1% glucose, 2% galactose medium shows a rise of UDP-glucose/-galactose epimerase activity while no detectable increase in activity is observed in the uge1Dgal10D strain. Gal10p can replace loss of Uge1p in the uge1DELTA mutant in glucose medium. Growth and galactosylation phenotypes of uge1DELTA, overview
additional information
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construction of disruption mutants, DELTAuge1 and DELTAuge1DELTAgal10. Both mutant strains are sensitive to hygromycin B. Oligosaccharide content is reduced in acid phosphatase prepared from the uge1DELTA strain. The uge1DELTA strain grown in 0.1% glucose, 2% galactose medium shows a rise of UDP-glucose/-galactose epimerase activity while no detectable increase in activity is observed in the uge1Dgal10D strain. Gal10p can replace loss of Uge1p in the uge1DELTA mutant in glucose medium. Growth and galactosylation phenotypes of uge1DELTA, overview
additional information
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construction of disruption mutants, DELTAuge1 and DELTAuge1DELTAgal10. Both mutant strains are sensitive to hygromycin B. Oligosaccharide content is reduced in acid phosphatase prepared from the uge1DELTA strain. The uge1DELTA strain grown in 0.1% glucose, 2% galactose medium shows a rise of UDP-glucose/-galactose epimerase activity while no detectable increase in activity is observed in the uge1Dgal10D strain. Gal10p can replace loss of Uge1p in the uge1DELTA mutant in glucose medium. Growth and galactosylation phenotypes of uge1DELTA, overview
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