1.1.1.22 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 760597 1.1.1.22 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 760583 1.1.1.22 A222Q/S233G is a dimer in solution 695880 1.1.1.22 A222Q/S233G mutation does not affect expression, stability, and secondary structure. Mutant protein is a dimer and catalytic active, with increased Km values for substrates 687474 1.1.1.22 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 761985 1.1.1.22 C260A 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 761284 1.1.1.22 C260A 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 655477 1.1.1.22 C276A is a hexamer-dimer mixture 695880 1.1.1.22 C276A site-directed mutagenesis, strong decrease in specific activity 685143 1.1.1.22 C276E activity is not measurable at pH 8.7, 22°C 667848 1.1.1.22 C276G activity is not measurable at pH 8.7, 22°C 667848 1.1.1.22 C276K activity is not measurable at pH 8.7, 22°C 667848 1.1.1.22 C276L activity is not measurable at pH 8.7, 22°C 667848 1.1.1.22 C276S is a hexamer 695880 1.1.1.22 C276S no enzymic activity, affinity for NAD+ similar to wild-type, retains predominantly hexameric structure 656220 1.1.1.22 C276S site-directed mutagenesis, strong decrease in specific activity 685143 1.1.1.22 C276Y activity is not measurable at pH 8.7, 22°C 667848 1.1.1.22 D280A extremely poor enzymic activity 725478 1.1.1.22 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 685143 1.1.1.22 D280N an inactive UGDH mutant 711665 1.1.1.22 D280N shows, exclusively, a hexameric quaternary structure in solution 695880 1.1.1.22 D280N site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function 685143 1.1.1.22 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 724328 1.1.1.22 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 740715 1.1.1.22 E141Q kcat-value 10fold lower than wild-type 655477 1.1.1.22 E145Q kcat-value 10fold lower than wild-type 655477 1.1.1.22 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 725478 1.1.1.22 G13E normal expression and stability of mutant, no enzymic activity, no photoaffinity labeling with nicotinamide 2-azidoadenosine dinucleotide 656240 1.1.1.22 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 761985 1.1.1.22 K220A extremely poor enzymic activity 725478 1.1.1.22 K220A shows, exclusively, a hexameric quaternary structure in solution 695880 1.1.1.22 K220A site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function 685143 1.1.1.22 K220H site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function 685143 1.1.1.22 K220R site-directed mutagenesis, putative active site residue, mutation severly impairs enzyme function 685143 1.1.1.22 K279A is essentially a dimer 695880 1.1.1.22 K279A no enzymic activity, affinity for NAD+ similar to wild-type, almost exclusively found as dimer 656220 1.1.1.22 K279A site-directed mutagenesis, strong decrease in specific activity 685143 1.1.1.22 K323A site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type enzyme 740710 1.1.1.22 K339A is a dimer 695880 1.1.1.22 K339A site-directed mutagenesis, 165fold decrease in affinity for UDP-glucose. Mutant forms a dimer, in contrast to hexameric wild-type 685143 1.1.1.22 K94E mutation in the hexamer-building interface, generates a stable enzyme dimer. 160fold decrease in kcat value 724364 1.1.1.22 K94E substitution prevents hexamer formation. Mutant does not display hysteresis 724370 1.1.1.22 additional information comparison of sequence homologies with bacterial enzymes 286306 1.1.1.22 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 710889 1.1.1.22 additional information differences in host colonization between wild-type and UDPDH mutant 698005 1.1.1.22 additional information enzyme Ugd from Escherichia coli K-12 can functionally replace enzyme Ugd from Escherichia coli serotype K30 in biosynthesis of K30 capsular polysaccharide 740710 1.1.1.22 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) 700059 1.1.1.22 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 760577 1.1.1.22 additional information mutant lacking PA2022 activity and double mutant lacking PA2022 and isoform PA3559 activity are more susceptible to chloramphenicol, ceffotaxime, and ampicillin 687559 1.1.1.22 additional information mutant lacking PA3559 activity shows reduced resistance to polymyxin B 687559 1.1.1.22 additional information overexpression of enzyme plus transformation of gene cluster for K5 polysaccharide production, 15fold increase in enzyme activity, decrease in K5 polysaccharide formation 654528 1.1.1.22 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 -, 739891 1.1.1.22 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 695880 1.1.1.22 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 740715 1.1.1.22 additional information UGDH overexpression stimulates hyaluronan production in HEK293 cells 711665 1.1.1.22 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 739967 1.1.1.22 N224A steady-state kinetic parameters are within an order of magnitude of the native enzyme 725478 1.1.1.22 R324A site-directed mutagenesis, the mutant purifies in much lower amounts relative to wild-type and is prone to degradation and has negligible activity 740710 1.1.1.22 T118A 160fold reduction of kcat value 655477 1.1.1.22 T131S steady-state kinetic parameters are within an order of magnitude of the native enzyme 725478 1.1.1.22 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 740715 1.1.1.22 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 740715 1.1.1.22 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 725260 1.1.1.22 Y10K mutation in GXGYXG consensus motif, 2% residual activity 725260 1.1.1.22 Y10S mutation in GXGYXG consensus motif, 3% residual activity 725260 1.1.1.22 Y71F site-directed mutagenesis, the mutant shows unaltered catalytic activity 740710