5.1.3.14 D413K enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity 641739 5.1.3.14 D413N enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity 641739 5.1.3.14 H110A mutant enzyme shows a drastic loss of epimerase activity, oligomerization is significantly different from that of the wild-type enzyme,loss of epimerase activity can largely by attributed to incorrect protein folding 641739 5.1.3.14 H132A mutant enzyme shows a drastic loss of epimerase activity, oligomerization is significantly different from that of the wild-type enzyme, loss of epimerase activity can largely by attributed to incorrect protein folding 641739 5.1.3.14 H155A mutant enzyme forms mainly trimeric enzyme with small amounts of hexamer 641739 5.1.3.14 H155A mutant enzyme shows a drastic loss of epimerase activity, loss of epimerase activity can largely by attributed to incorrect protein folding 641739 5.1.3.14 H157A mutant enzyme forms mainly trimeric enzyme with small amounts of hexamer 641739 5.1.3.14 H157A mutant enzyme shows a drastic loss of epimerase activity, loss of epimerase activity can largely by attributed to incorrect protein folding 641739 5.1.3.14 H45A mutant enzyme shows a drastic loss of epimerase activity 641739 5.1.3.14 R420M enzyme with mutation in the putative kinase active site shows drastic loss in their kinase activity but retains their epimerase activity 641739 5.1.3.14 DELTA1-234 mutant enzyme shows no N-epimerase activity 660988 5.1.3.14 DELTA1-356 mutant enzyme shows no N-epimerase activity 660988 5.1.3.14 DELTA1-39 mutant enzyme shows no N-epimerase activity 660988 5.1.3.14 DELTA383-722 epimerase activity is 2% of wild-type enzyme 660988 5.1.3.14 DELTA490-722 epimerase activity is 15% of wild-type enzyme 660988 5.1.3.14 DELTA597-722 epimerase activity is 2% of wild-type enzyme 660988 5.1.3.14 DELTA697-722 epimerase activity is about 70% of wild-type enzyme 660988 5.1.3.14 DELTA717-722 epimerase activity is about 95% of wild-type enzyme 660988 5.1.3.14 D100N no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine -, 661102 5.1.3.14 D131N no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis -, 661102 5.1.3.14 E122Q no conversion of UDP-N-acetyl-D-glucosamine to UDP + N-acetyl-D-mannosamine, acetamidoglucal is released from the active site during catalysis -, 661102 5.1.3.14 D95N about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants 661247 5.1.3.14 E117Q about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants 661247 5.1.3.14 E131Q about 18000 fold decrease in turnover number for UDP-N-acetyl-D-glucosamine, not possible to obtain accurate kinetic constants 661247 5.1.3.14 H213N 30fold increase in Km-value and 50fold decrease in turnover-number for UDP-N-acetyl-D-glucosamine. Unlike the wild-type enzyme no inhibition is detected at UDP-concentrations up to 10 mM 661247 5.1.3.14 K15A more than 100fold increase in KM-value for UDP-N-acetyl-D-glucosamine 661247 5.1.3.14 M712T the homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy 661735 5.1.3.14 G135V/R246W mutation in patients with hereditary inclusion body myopathy: G135V/R246W (GNE/GNE domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 38% of wild-type, N-acetylmannosamine kinase activity is 72% of wild-type 661840 5.1.3.14 M712T/M712T M712T/M712T (MNK/MNK domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 83% of wild-type, N-acetylmannosamine kinase activity is 55% of wild-type 661840 5.1.3.14 V216A/A631V V216A/A631V (GNE/MNK domain mutation), UDP-N-acetylglucosamine 2-epimerase activity is 48% of wild-type, N-acetylmannosamine kinase activity is 63% of wild-type 661840 5.1.3.14 A630T mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 70-80% of wild-type activity 662186 5.1.3.14 C13S mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 D176V mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 D177C mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 D378Y mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 G708S mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 50% of wild-type activity 662186 5.1.3.14 H132Q mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 I472T mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 50% of wild-type activity 662186 5.1.3.14 V331A mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to less than 20% of wild-type 662186 5.1.3.14 V572L mutation in patients with distal myopathy with rimmed vacuoles, UDP-N-acetylglucosamine 2-epimerase activity of mutant enzyme is reduced to 70-80% of wild-type activity 662186 5.1.3.14 C303V exhibited almost no reduction in epimerase activity 672131 5.1.3.14 C303X the C303X protein does not display any enzymatic activity 672131 5.1.3.14 D378Y 60% reduction of epimerase activity 672131 5.1.3.14 I200F exhibited almost no reduction in epimerase activity 672131 5.1.3.14 R266Q GNE mutants are created by site-directed mutagenesis with the mutagenic oligonucleotides 5'-GGTTCGAGTGATGCAGAAGAAGGGCATTGAGCA-3' for the R266Q sialuria mutations (where the site of mutation is underlined) through PCR-like amplification with Pfu polymerase. 674638 5.1.3.14 R266W GNE mutants are created by site-directed mutagenesis with the mutagenic oligonucleotides 5'-GGTTCGAGTGATGTGGAAGAAGGGCATTGAGCA-3' for the R266W sialuria mutations (where the site of mutation is underlined) through PCR-like amplification with Pfu polymerase. 674638 5.1.3.14 additional information transgenic Arabidopsis thaliana plants expressing three key enzymes of the mammalian Neu5Ac biosynthesis pathway: UDPN-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, N-acetylneuraminic acid phosphate synthase, and CMP-Nacetylneuraminic acid synthetase. Simultaneous expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase and N-acetylneuraminic acid phosphate synthase results in the generation of significant Neu5Ac amounts of 1275 nmol per g fresh weight in leaves, which can be further converted to cytidine monophospho-N-acetylneuraminic acid by coexpression of CMP-N-acetylneuraminic acid synthetase 689622 5.1.3.14 H242A dramatic decrease in catalytic efficiency 692076 5.1.3.14 H44Q dramatic decrease in catalytic efficiency 692076 5.1.3.14 Q43A dramatic decrease in catalytic efficiency 692076 5.1.3.14 Q70A dramatic decrease in catalytic efficiency 692076 5.1.3.14 R210A dramatic decrease in catalytic efficiency 692076 5.1.3.14 additional information splice variant hGNE2, recombinantly expressed in insect and mamalian cells, displays selective reduction of UDPGlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. Splice variant hGNE3 only possesses kinase activity 692536 5.1.3.14 additional information generation of GNE knockout mice by gene targeting, enzyme knockout leads to embryonic lethality, phenotype, overview. Impaired sialylation of glycoconjugates induces cell death, either by the loss of the sialic acid specific masking of cells to prevent proteolytic attack or by the prevention of cell migration and differentiation 702507 5.1.3.14 additional information sialuria is caused by the loss of feedback control of UDP-GlcNAc 2-epimerase activity due to the mutation of only one of the two arginine residues 263 and 266 702507 5.1.3.14 additional information the frame shif mutation 1295delA, leading to a premature stop codon at K432, is involved in hereditary inclusion body myopathy, phenotype, overview 703285 5.1.3.14 V696M naturally occuring missense mutation G2086A involved in hereditary inclusion body myopathy, phenotype, overview 703285 5.1.3.14 A631V a naturally occuring missense mutation in exon 11 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 E2G a naturally occuring missense mutation in exon 2 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 I142T a naturally occuring missense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 I298T a naturally occuring missense mutation in exon 5 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 L556S a naturally occuring missense mutation in exon 10 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 additional information a synonymous variation, p.Y591Y, codon tac>tat, is seen in a patient bearing compound heterozygous nonsynonymous mutation, p.S615X and p.Y675H 703865 5.1.3.14 Q436X a naturally occuring nonsense mutation in exon 8 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 R246Q a naturally occuring missense mutation in exon 4 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 R335W a naturally occuring missense mutation in exon 6 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 R71W a naturally occuring missense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 R8X a naturally occuring nonsense mutation in exon 2 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 S615X a naturally occuring nonsense mutation in exon 11 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 V696M a naturally occuring missense mutation in exon 12 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 W204X a naturally occuring nonsense mutation in exon 3 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 Y675H a naturally occuring missense mutation in exon 12 of the GNE gene of a patient with hereditary inclusion body myopathy 703865 5.1.3.14 C13S a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 C303V a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 C303X a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 D176V a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 D225N a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 D378Y a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 G135V a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 G206S a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 G89R a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 H132Q a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 I200F a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 I241S a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 L379H a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 M171V a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 M29T a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 M712T the Persian-Jewish HIBM founder mutation is located at the interface alpha4alpha10 of GNE and likely affects GlcNAc, Mg2+, and ATP binding 703912 5.1.3.14 additional information mutant genotyping, overview. Modeling of effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action, overview. All reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK 703912 5.1.3.14 P27S a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 P283S a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 P36L a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R11W a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R129Q a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R162C a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R177C a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R202L a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R246Q a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R246W a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R263L a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R266Q a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R266W a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R277C a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R306Q a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 R335W a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 V216A a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 V331A a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 V367I a naturally occuirng missense mutation the epimerase part of the bifunctional enzyme 703912 5.1.3.14 additional information gene mnaA, genotyping and mutation identification. Mapping of MnaA LOF mutations into the MnaA crystal structure revealing key residues for substrate binding site stability and charge. To determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects. Complementing DELTAcap5P mnaASa P12L and DELTAcap5P mnaASa Y194* with either cap5P or mnaASa reintroduced on an inducible plasmid restores WTA polymer levels, resistance to each of the beta-lactams tested, and wild-type sensitivity to L638 -, 755205 5.1.3.14 additional information gene mnaA, genotyping and mutation identification. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects -, 755205 5.1.3.14 P12L site-diected mutagenesis -, 755205 5.1.3.14 additional information to determine whether L638R mnaA LOF mutants are not identified in MRSA COL due to a functional redundancy between Cap5P and MnaA, a cap5P deletion mutant is constructed and the L638R studies are repeated. Under these conditions, in addition to identifying the expected tarG L638R mutations as well as tarO and tarA LOF mutations, multiple (n = 11) independent resistor isolates obtained uniquely possess distinct mutations that map to mnaA and are predicted to inactivate gene function as well as directly confer L638R drug resistance based on the absence of additional non-synonymous mutations in their genome following WGS analysis. While MRSA COL DELTAcap5P exhibits no wall teichoic acid (WTA) depletion phenotype and remains resistant to beta-lactams, MRSA COL mnaA, DELTAcap5P double mutants are completely devoid of WTA and are also highly sensitive to beta-lactams. MRSA COL mnaA, cap5P double mutants and MRSE mnaA single mutants reveal morphological phenotypes consistent with WTA depletion, including increased cell size heterogeneity and septation defects -, 755205 5.1.3.14 Y194X site-directed mutagenesis -, 755205