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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
I331D
hypomorphic loss-of-function mutation jekyll m151. contrary to humans, homozygous mutant larvae do not show signs of increased c-fos expression at basal state
K323A
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site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme
R324A
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site-directed mutagenesis, the mutant purifies in much lower amounts relative to wild-type and is prone to degradation and has negligible activity
Y71F
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site-directed mutagenesis, the mutant shows unaltered catalytic activity
A104L
ubstitution introduced to fill a cavity in the E state and sterically prevent repacking of the core into the inactive Eomega state. Mutant A104L does not show hysteresis or negative cooperativity, binds UDP-xylose with lower affinity and the inhibition is no longer cooperative
A136M
mutant does not exhibit substrate cooperativity. The inhibitor affinity of A136M is reduced 14fold and does not exhibit hysteresis. Substitution disrupts NAD+-induced negative cooperativity
A44V
mutation is the genetic cause of a developmental epileptic encephalopathy in a consanguineous Palestinian family with three affected siblings. The A44V variant is also found in two additional families from Puerto Rico and from Spain
C276E
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activity is not measurable at pH 8.7, 22°C
C276G
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activity is not measurable at pH 8.7, 22°C
C276K
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activity is not measurable at pH 8.7, 22°C
C276L
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activity is not measurable at pH 8.7, 22°C
C276Y
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activity is not measurable at pH 8.7, 22°C
D280A
extremely poor enzymic activity
D280E
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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
E110A
site-directed mutagenesis, the mutant, although dimeric in the apo form, exhibits only about 50% reduction in Vmax, but is highly unstable in solution and in cultured cells so it cannot be evaluated unambiguously
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
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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
T325A
site-directed mutagenesis, the mutant occurs as dimeric species that can be induced to form hexamers in the ternary complex with substrate and cofactor. The inducible hexamer shows that upon increasing enzyme concentration, which favors the hexameric species, activity is modestly decreased and exhibits cooperativity. The T325A mutant is significantly less efficient in promoting downstream hyaluronan production by HEK293 cells than the wild-type. The activity of the T325A mutant is the most labile, with a half-life of only 24 h that is not extended significantly by substrate and cofactor addition
T325D
site-directed mutagenesis, the mutant yields exclusively dimeric species. The T325D mutant is significantly less efficient in promoting downstream hyaluronan production by HEK293 cells than the wild-type. UGDH T325D retains its activity similarly to the wild-type enzyme but does not exhibit increased stability in the abortive ternary complex
E141Q
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kcat-value 10fold lower than wild-type
E145Q
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kcat-value 10fold lower than wild-type
T118A
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160fold reduction of kcat value
A222Q/S233G
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mutation does not affect expression, stability, and secondary structure. Mutant protein is a dimer and catalytic active, with increased Km values for substrates
A222Q/S233G
is a dimer in solution
C276A
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site-directed mutagenesis, strong decrease in specific activity
C276A
is a hexamer-dimer mixture
C276S
no enzymic activity, affinity for NAD+ similar to wild-type, retains predominantly hexameric structure
C276S
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site-directed mutagenesis, strong decrease in specific activity
D280N
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site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function
D280N
shows, exclusively, a hexameric quaternary structure in solution
D280N
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an inactive UGDH mutant
K220A
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site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function
K220A
shows, exclusively, a hexameric quaternary structure in solution
K220A
extremely poor enzymic activity
K279A
no enzymic activity, affinity for NAD+ similar to wild-type, almost exclusively found as dimer
K279A
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site-directed mutagenesis, strong decrease in specific activity
K279A
is essentially a dimer
K339A
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site-directed mutagenesis, 165fold decrease in affinity for UDP-glucose. Mutant forms a dimer, in contrast to hexameric wild-type
K94E
mutation in the hexamer-building interface, generates a stable enzyme dimer. 160fold decrease in kcat value
K94E
substitution prevents hexamer formation. Mutant does not display hysteresis
C260A
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mutation of the essential Cys residue. The C260A mutant and wild-type are then co-expressed in vivo via a single-crossover homologous recombination method. The resulting strain produces an amide derivative of hyaluronan
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
additional information
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differences in host colonization between wild-type and UDPDH mutant
additional information
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comparison of sequence homologies with bacterial enzymes
additional information
inactivation of gene Ugd(BCAL2946) results in increased sensitivity to polymyxin B and this sensitivity can be overcome when either genes Ugd(BCAL2946) or Ugd(BCAM0855) but not gene ugd(BCAM2034) is expressed from plasmids. Growth of a conditional Ugd(BCAL2946) mutant, created in the DELTAUgd(BCAM0855) background, is significantly impaired under non-permissive conditions. Growth can be rescued by either Ugd(BCAL2946) or Ugd(BCAM0855) expressed in trans, but not by Ugd(BCAM2034)
additional information
inactivation of gene Ugd(BCAL2946) results in increased sensitivity to polymyxin B and this sensitivity can be overcome when either genes Ugd(BCAL2946) or Ugd(BCAM0855) but not gene ugd(BCAM2034) is expressed from plasmids. Growth of a conditional Ugd(BCAL2946) mutant, created in the DELTAUgd(BCAM0855) background, is significantly impaired under non-permissive conditions. Growth can be rescued by either Ugd(BCAL2946) or Ugd(BCAM0855) expressed in trans, but not by Ugd(BCAM2034)
additional information
inactivation of gene Ugd(BCAL2946) results in increased sensitivity to polymyxin B and this sensitivity can be overcome when either genes Ugd(BCAL2946) or Ugd(BCAM0855) but not gene ugd(BCAM2034) is expressed from plasmids. Growth of a conditional Ugd(BCAL2946) mutant, created in the DELTAUgd(BCAM0855) background, is significantly impaired under non-permissive conditions. Growth can be rescued by either Ugd(BCAL2946) or Ugd(BCAM0855) expressed in trans, but not by Ugd(BCAM2034)
additional information
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overexpression of enzyme plus transformation of gene cluster for K5 polysaccharide production, 15fold increase in enzyme activity, decrease in K5 polysaccharide formation
additional information
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enzyme Ugd from Escherichia coli K-12 can functionally replace enzyme Ugd from Escherichia coli serotype K30 in biosynthesis of K30 capsular polysaccharide
additional information
perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. Structural stability at the dimeric interface is necessary to retain the hexameric structure of UGDH
additional information
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perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. Structural stability at the dimeric interface is necessary to retain the hexameric structure of UGDH
additional information
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UGDH overexpression stimulates hyaluronan production in HEK293 cells
additional information
UDP-glucose dehydrogenase mutants are engineered to perturb hexamer:dimer quaternary structure equilibrium. Dimeric species of UGDH have reduced activity in vitro and in supporting hyaluronan production by cultured cells. The purified enzymes reveal a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. The activity of the truncated DELTA132 mutant is negligible. The half-life of UGDH catalytic activity in vitro is reduced by mutations at the dimer interface
additional information
UGDH specific siRNAs markedly inhibits UGDH mRNA and protein expression, and leads to an obvious suppression of PGs synthesis in human articular chondrocytes
additional information
introduction of site-specific unnatural amino acids to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking is achieved by encoding 4-benzoyl-L-phenylalanine at position 458, and exposing to long wavelength UV in the presence of substrate and cofactor. Purified hexameric complexes contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme is reduced by almost 60% relative to the uncrosslinked UGDH mutant
additional information
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introduction of site-specific unnatural amino acids to facilitate crosslinking of monomeric subunits into predominantly obligate oligomeric species. Optimal crosslinking is achieved by encoding 4-benzoyl-L-phenylalanine at position 458, and exposing to long wavelength UV in the presence of substrate and cofactor. Purified hexameric complexes contain significant fractions of dimer and trimer (approximately 50%) along with another 10% tetramer and higher molecular mass species. Activity of the crosslinked enzyme is reduced by almost 60% relative to the uncrosslinked UGDH mutant
additional information
construction by Tn5 transposon mutagenesis of a knockout mutant of ugd, that is extremely sensitive to polymyxin B, presumably because of alterations in lipopolysaccharide structure and cell surface architecture in the mutant. The mutant is defective in swarming, expresses lower levels of virulence factor hemolysin, and has lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd gene leads to the restoration of wild-type phenotypic traits, phenotype, overview
additional information
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construction by Tn5 transposon mutagenesis of a knockout mutant of ugd, that is extremely sensitive to polymyxin B, presumably because of alterations in lipopolysaccharide structure and cell surface architecture in the mutant. The mutant is defective in swarming, expresses lower levels of virulence factor hemolysin, and has lower cell invasion ability. Complementation of the ugd or galU mutant with the full-length ugd gene leads to the restoration of wild-type phenotypic traits, phenotype, overview
additional information
mutant lacking PA2022 activity and double mutant lacking PA2022 and isoform PA3559 activity are more susceptible to chloramphenicol, ceffotaxime, and ampicillin
additional information
mutant lacking PA2022 activity and double mutant lacking PA2022 and isoform PA3559 activity are more susceptible to chloramphenicol, ceffotaxime, and ampicillin
additional information
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mutant lacking PA2022 activity and double mutant lacking PA2022 and isoform PA3559 activity are more susceptible to chloramphenicol, ceffotaxime, and ampicillin
additional information
mutant lacking PA3559 activity shows reduced resistance to polymyxin B
additional information
mutant lacking PA3559 activity shows reduced resistance to polymyxin B
additional information
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mutant lacking PA3559 activity shows reduced resistance to polymyxin B
additional information
overexpression of the ugdG gene in Sphingomonas sanxanigenens results in increased sphingan Ss production and higher fermentation broth viscosity. The weightaverage molecular weight of polymer Ss from the recombinant strain is higher and the viscosity is higher than those from the wild-type strain at a shear rate of 1 rev/min
additional information
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overexpression of the ugdG gene in Sphingomonas sanxanigenens results in increased sphingan Ss production and higher fermentation broth viscosity. The weightaverage molecular weight of polymer Ss from the recombinant strain is higher and the viscosity is higher than those from the wild-type strain at a shear rate of 1 rev/min
additional information
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overexpression of the ugdG gene in Sphingomonas sanxanigenens results in increased sphingan Ss production and higher fermentation broth viscosity. The weightaverage molecular weight of polymer Ss from the recombinant strain is higher and the viscosity is higher than those from the wild-type strain at a shear rate of 1 rev/min
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