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Werner Reutter - One of the best experts on this subject based on the ideXlab platform.
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Selective Loss of either the Epimerase or Kinase Activity of UDP-N-acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase due to Site-directed Mutagenesis Based on Sequence Alignments
Journal of Biological Chemistry, 1999Co-Authors: Karin Effertz, Stephan Hinderlich, Werner ReutterAbstract:Abstract N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifunctional enzyme displays sequence similarities with prokaryotic UDP-GlcNAc 2-Epimerases, whereas the sequence of its C-terminal region is similar to sequences of members of the sugar kinase superfamily. High level overexpression of active enzyme was established by using the baculovirus/Sf9 system. For functional characterization, site-directed mutagenesis was performed on different conserved amino acid residues. The histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of Epimerase activity with almost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their Epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were shown to form a hexamer like the wild-type enzyme, indicating little influence of mutation on protein folding. Histidine mutants H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer. Oligomerization of mutants H110A and H132A was also significantly different from that of the wild-type enzyme. Therefore the loss of Epimerase activity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A seems to be involved directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase are essential for the phosphorylation process. The fact that either Epimerase or kinase activity are lost selectively provides evidence for the existence of two active sites working quite independently.
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selective loss of either the Epimerase or kinase activity of udp n acetylglucosamine 2 Epimerase n acetylmannosamine kinase due to site directed mutagenesis based on sequence alignments
Journal of Biological Chemistry, 1999Co-Authors: Karin Effertz, Stephan Hinderlich, Werner ReutterAbstract:N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifunctional enzyme displays sequence similarities with prokaryotic UDP-GlcNAc 2-Epimerases, whereas the sequence of its C-terminal region is similar to sequences of members of the sugar kinase superfamily. High level overexpression of active enzyme was established by using the baculovirus/Sf9 system. For functional characterization, site-directed mutagenesis was performed on different conserved amino acid residues. The histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of Epimerase activity with almost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their Epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were shown to form a hexamer like the wild-type enzyme, indicating little influence of mutation on protein folding. Histidine mutants H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer. Oligomerization of mutants H110A and H132A was also significantly different from that of the wild-type enzyme. Therefore the loss of Epimerase activity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A seems to be involved directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase are essential for the phosphorylation process. The fact that either Epimerase or kinase activity are lost selectively provides evidence for the existence of two active sites working quite independently.
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UDP-GlcNAc 2-Epimerase: A Regulator of Cell Surface Sialylation
Science, 1999Co-Authors: Oliver T Keppler, Josmar Langner, Reinhard Schwartz-albiez, Stephan Hinderlich, Werner Reutter, Michael PawlitaAbstract:Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-Epimerase (UDP-GlcNAc 2-Epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-Epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.
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A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-Epimerase/N-ACETYLMANNOSAMINE KINASE
Journal of Biological Chemistry, 1997Co-Authors: Roger Stäsche, Stephan Hinderlich, Karin Effertz, Lothar Lucka, Christoph Weise, Petra Moormann, Werner ReutterAbstract:N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-Epimerase (UDP-GlcNAc 2-Epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stasche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313-24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both Epimerase and kinase activity. Distribution of UDP-GlcNAc 2-Epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-Epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-Epimerase/ManNAc kinase mRNA in the non-secreting tissues.
Albert M Berghuis - One of the best experts on this subject based on the ideXlab platform.
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overlapping and distinct roles of aspergillus fumigatus udp glucose 4 Epimerases in galactose metabolism and the synthesis of galactose containing cell wall polysaccharides
Journal of Biological Chemistry, 2014Co-Authors: Fabrice N Gravelat, Stefanie D Baptista, Paolo Campoli, Sein Choe, Ilia Kravtsov, Carole Creuzenet, Albert M Berghuis, Evgeny Vinogradov, Robert P. Cerone, Jean-paul LatgéAbstract:The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-Epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these Epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-Epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose Epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.
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a single bifunctional udp glcnac glc 4 Epimerase supports the synthesis of three cell surface glycoconjugates in campylobacter jejuni
Journal of Biological Chemistry, 2005Co-Authors: Stephane Bernatchez, Albert M Berghuis, Noboru Ishiyama, Christine M Szymanski, Harold C Jarrell, Peter C Y Lau, Martin N Young, Warren W WakarchukAbstract:Abstract The major cell-surface carbohydrates (lipooligosaccharide, capsule, and glycoprotein N-linked heptasaccharide) of Campylobacter jejuni NCTC 11168 contain Gal and/or GalNAc residues. GalE is the sole annotated UDP-glucose 4-Epimerase in this bacterium. The presence of GalNAc residues in these carbohydrates suggested that GalE might be a UDP-GlcNAc 4-Epimerase. GalE was shown to epimerize UDP-Glc and UDP-GlcNAc in coupled assays with C. jejuni glycosyltransferases and in sugar nucleotide epimerization equilibria studies. Thus, GalE possesses UDP-GlcNAc 4-Epimerase activity and was renamed Gne. The K m(app) values of a purified MalE-Gne fusion protein for UDP-GlcNAc and UDP-GalNAc are 1087 and 1070 μm, whereas those for UDP-Glc and UDP-Gal are 780 and 784 μm. The kcat and kcat/K m(app) values were three to four times higher for UDP-GalNAc and UDP-Gal than for UDP-GlcNAc and UDP-Glc. The comparison of the kinetic parameters of MalE-Gne to those of other characterized bacterial UDP-GlcNAc 4-Epimerases indicated that Gne is a bifunctional UDP-GlcNAc/Glc 4-Epimerase. The UDP sugar-binding site of Gne was modeled by using the structure of the UDP-GlcNAc 4-Epimerase WbpP from Pseudomonas aeruginosa. Small differences were noted, and these may explain the bifunctional character of the C. jejuni Gne. In a gne mutant of C. jejuni, the lipooligosaccharide was shown by capillary electrophoresis-mass spectrometry to be truncated by at least five sugars. Furthermore, both the glycoprotein N-linked heptasaccharide and capsule were no longer detectable by high resolution magic angle spinning NMR. These data indicate that Gne is the enzyme providing Gal and GalNAc residues with the synthesis of all three cell-surface carbohydrates in C. jejuni NCTC 11168.
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Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-Epimerase from Pseudomonas aeruginosa: substrate specificity in udp-hexose 4-Epimerases.
The Journal of biological chemistry, 2004Co-Authors: Noboru Ishiyama, Carole Creuzenet, Joseph S. Lam, Albert M BerghuisAbstract:Abstract The O antigen of lipopolysaccharide in Gram-negative bacteria plays a critical role in bacterium-host interactions, and for pathogenic bacteria it is a major virulence factor. In Pseudomonas aeruginosa serotype O6 one of the initial steps in O-antigen biosynthesis is catalyzed by a saccharide Epimerase, WbpP. WbpP is a member of the UDP-hexose 4-Epimerase family of enzymes and exists as a homo-dimer. This enzyme preferentially catalyzes the conversion between UDP-GlcNAc and UDPGalNAc above UDP-Glc and UDP-Gal, using NAD+ as a cofactor. The crystal structures of WbpP in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 Ǎ, respectively, which represents the first structural studies of a genuine UDP-GlcNAc 4-Epimerase. These structures in combination with complementary mutagenesis studies suggest that the basis for the differential substrate specificity of WbpP is a consequence of the presence of a pliable solvent network in the active site. This information allows for a comprehensive analysis of the relationship between sequence and substrate specificity for UDP-hexose 4-Epimerases and enables the formulation of consensus sequences that predict substrate specificity of UDP-hexose 4-Epimerases yet to be biochemically characterized. Furthermore, the examination indicates that as little as one residue can dictate substrate specificity. Nonetheless, phylogenetic analysis suggests that this substrate specificity is an evolutionary and highly conserved property within UDP-hexose 4-Epimerases.
Amar Bhaduri - One of the best experts on this subject based on the ideXlab platform.
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UDPgalactose 4-Epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-Epimerase activity
European journal of biochemistry, 2004Co-Authors: Siddhartha Majumdar, Jhuma Ghatak, Sucheta Mukherji, Hiranmoy Bhattacharjee, Amar BhaduriAbstract:UDPgalactose 4-Epimerase (Epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae Epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast Epimerase has an N-terminal domain (residues 1–377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-Epimerase, and a C-terminal domain (residues 378–699), which shows extensive identity to either the bacterial or human aldose 1-Epimerase (mutarotase). The S. cerevisiae Epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified Epimerase preparations showed mutarotase activity and could convert either α-d-glucose or α-d-galactose to their β-anomers. Induction of cells with galactose led to simultaneous enhancement of both Epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast Epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5′-UMP and l-arabinose, Epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-Epimerase is a bifunctional enzyme with aldose 1-Epimerase activity. The active sites for these two enzymatic activities are located in different regions of the Epimerase holoenzyme.
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A bifunctional enzyme with aldose 1-Epimerase activity
2004Co-Authors: Siddhartha Majumdar, Jhuma Ghatak, Sucheta Mukherji, Hiranmoy Bhattacharjee, Amar BhaduriAbstract:UDPgalactose 4-Epimerase (Epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme ofthe galactose metabolic pathway. The Saccharomyces cerevisiae Epimerase encoded by the GAL10 gene is about twice the size ofeither the bacterial or human protein. Sequence analysis indicates that the yeast Epimerase has an N-terminal domain (residues 1–377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-Epimerase, and a C-terminal domain (residues 378–699), which shows extensive identity to either the bacterial or human aldose 1-Epimerase (mutarotase). The S. cerevisiae Epimerase was purified to > 95% homogeneity by sequential chromatography on DEAESephacel and Resource-Q columns. Purified Epimerase preparations showed mutarotase activity and could convert either a-D-glucose or a-D-galactose to their b-anomers. Induction ofcells with galactose led to simultaneous enhancement ofboth Epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property ofthe yeast Epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5¢-UMP and L-arabinose, Epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-Epimerase is a bifunctional enzyme with aldose 1-Epimerase activity. The active sites for these two enzymatic activities are located in different regions ofthe Epimerase holoenzyme.
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Two tryptophans at the active site of UDP-glucose 4-Epimerase from Kluyveromyces fragilis.
The Journal of biological chemistry, 1995Co-Authors: Sangeeta Ray, Sucheta Mukherji, Amar BhaduriAbstract:Abstract Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-Epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native Epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in Epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in Epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated Epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified Epimerase, complemented by a linear Stern-Volmer plot (effective Stern- Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.
Svein Valla - One of the best experts on this subject based on the ideXlab platform.
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structural and mutational characterization of the catalytic a module of the mannuronan c 5 Epimerase alge4 from azotobacter vinelandii
Journal of Biological Chemistry, 2008Co-Authors: Henriette J Rozeboom, Svein Valla, Helga Ertesvag, Tonje M Bjerkan, Kor H Kalk, Synnove Holtan, Finn Lillelund Aachmann, Bauke W DijkstraAbstract:Abstract Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic acid and its C-5 epimer α-l-guluronic acid. The polymer is first produced as polymannuronic acid and the guluronic acid residues are then introduced at the polymer level by mannuronan C-5-Epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-Epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction.
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the pseudomonas fluorescens algg protein but not its mannuronan c 5 Epimerase activity is needed for alginate polymer formation
Journal of Bacteriology, 2003Co-Authors: Martin Gimmestad, Helga Ertesvag, Gudmund Skjakbraek, Havard Sletta, Karianne Bakkevig, Sumita Jain, Sangjin Suh, Trond E Ellingsen, Dennis E Ohman, Svein VallaAbstract:Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-Epimerase AlgG. All mutations affected amino acids conserved among AlgG-Epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted Epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an Epimerase-defective (point mutation) and a wild-type Epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.
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Mannuronan C‐5 Epimerases and cellular differentiation of Azotobacter vinelandii
Environmental Microbiology, 2000Co-Authors: Hilde Kristin Hoidal, Martin Gimmestad, Britt Iren Glaerum Svanem, Svein VallaAbstract:Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a co-polymer of β- d-mannuronic acid (M) and α- l-guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 Epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 Epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the Epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the Epimerase-encoded R-modules. This gene is located within the Epimerase gene cluster of Azotobacter vinelandii.
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Mannuronan C-5 Epimerases and cellular differentiation of Azotobacter vinelandii.
Environmental microbiology, 2000Co-Authors: Hilde Kristin Hoidal, Martin Gimmestad, Britt Iren Glaerum Svanem, Svein VallaAbstract:Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 Epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 Epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the Epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the Epimerase-encoded R-modules. This gene is located within the Epimerase gene cluster of Azotobacter vinelandii.
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cloning and expression of an azotobacter vinelandii mannuronan c 5 Epimerase gene
Journal of Bacteriology, 1994Co-Authors: Helga Ertesvag, Gudmund Skjakbraek, Berit Doseth, Bjorn Larsen, Svein VallaAbstract:An Azotobacter vinelandii mannuronan C-5-Epimerase gene was cloned in Escherichia coli. This enzyme catalyzes the Ca(2+)-dependent epimerization of D-mannuronic acid residues in alginate to the corresponding epimer L-guluronic acid. The Epimerase gene was identified by screening a bacteriophage EMBL3 gene library of A. vinelandii DNA with a synthetic oligonucleotide probe. The sequence of this probe was deduced after determination of the N-terminal amino acid sequence of a previously reported extracellular mannuronan C-5-Epimerase from A. vinelandii. A DNA fragment hybridizing against the probe was subcloned in a plasmid vector in E. coli, and the corresponding recombinant plasmid expressed intracellular mannuronan C-5-Epimerase in this host. The nucleotide sequence of the gene encoding the Epimerase was determined, and the sequence data showed that the molecular mass of the deduced protein is 103 kDa. A module consisting of about 150 amino acids was repeated tandemly four times in the C-terminal part of the deduced protein. Each of the four repeats contained four to six tandemly oriented nonameric repeats. The sequences in these motifs are similar to the Ca(2+)-binding domains of functionally unrelated secreted proteins reported previously in other bacteria. The reaction product of the recombinant Epimerase was analyzed by nuclear magnetic resonance spectroscopy, and the results showed that the guluronic acid residues were distributed in blocks along the polysaccharide chain. Such a nonrandom distribution pattern, which is important for the commercial use of alginate, has previously also been identified in the reaction product of the corresponding enzyme isolated from A. vinelandii.
Stephan Hinderlich - One of the best experts on this subject based on the ideXlab platform.
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Selective Loss of either the Epimerase or Kinase Activity of UDP-N-acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase due to Site-directed Mutagenesis Based on Sequence Alignments
Journal of Biological Chemistry, 1999Co-Authors: Karin Effertz, Stephan Hinderlich, Werner ReutterAbstract:Abstract N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifunctional enzyme displays sequence similarities with prokaryotic UDP-GlcNAc 2-Epimerases, whereas the sequence of its C-terminal region is similar to sequences of members of the sugar kinase superfamily. High level overexpression of active enzyme was established by using the baculovirus/Sf9 system. For functional characterization, site-directed mutagenesis was performed on different conserved amino acid residues. The histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of Epimerase activity with almost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their Epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were shown to form a hexamer like the wild-type enzyme, indicating little influence of mutation on protein folding. Histidine mutants H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer. Oligomerization of mutants H110A and H132A was also significantly different from that of the wild-type enzyme. Therefore the loss of Epimerase activity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A seems to be involved directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase are essential for the phosphorylation process. The fact that either Epimerase or kinase activity are lost selectively provides evidence for the existence of two active sites working quite independently.
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selective loss of either the Epimerase or kinase activity of udp n acetylglucosamine 2 Epimerase n acetylmannosamine kinase due to site directed mutagenesis based on sequence alignments
Journal of Biological Chemistry, 1999Co-Authors: Karin Effertz, Stephan Hinderlich, Werner ReutterAbstract:N-Acetylneuraminic acid is the most common naturally occurring sialic acid, as well as being the biosynthetic precursor of this group of compounds. UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase has been shown to be the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver, and it is a regulator of cell surface sialylation. The N-terminal region of this bifunctional enzyme displays sequence similarities with prokaryotic UDP-GlcNAc 2-Epimerases, whereas the sequence of its C-terminal region is similar to sequences of members of the sugar kinase superfamily. High level overexpression of active enzyme was established by using the baculovirus/Sf9 system. For functional characterization, site-directed mutagenesis was performed on different conserved amino acid residues. The histidine mutants H45A, H110A, H132A, H155A, and H157A showed a drastic loss of Epimerase activity with almost unchanged kinase activity. Conversely, the mutants D413N, D413K, and R420M in the putative kinase active site lost their kinase activity but retained their Epimerase activity. To estimate the structural perturbation effect due to site-directed mutagenesis, the oligomeric state of all mutants was determined by gel filtration analysis. The mutants D413N, D413K, and R420M as well as H45A were shown to form a hexamer like the wild-type enzyme, indicating little influence of mutation on protein folding. Histidine mutants H155A and H157A formed mainly trimeric enzyme with small amounts of hexamer. Oligomerization of mutants H110A and H132A was also significantly different from that of the wild-type enzyme. Therefore the loss of Epimerase activity in mutants H110A, H132A, H155A, and H157A can largely be attributed to incorrect protein folding. In contrast, the mutation site of mutant H45A seems to be involved directly in the epimerization process, and the amino acids Asp-413 and Arg-420 of UDP-GlcNAc 2-Epimerase/N-acetylmannosamine kinase are essential for the phosphorylation process. The fact that either Epimerase or kinase activity are lost selectively provides evidence for the existence of two active sites working quite independently.
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UDP-GlcNAc 2-Epimerase: A Regulator of Cell Surface Sialylation
Science, 1999Co-Authors: Oliver T Keppler, Josmar Langner, Reinhard Schwartz-albiez, Stephan Hinderlich, Werner Reutter, Michael PawlitaAbstract:Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-Epimerase (UDP-GlcNAc 2-Epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-Epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules.
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A Bifunctional Enzyme Catalyzes the First Two Steps in N-Acetylneuraminic Acid Biosynthesis of Rat Liver MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF UDP-N-ACETYL-GLUCOSAMINE 2-Epimerase/N-ACETYLMANNOSAMINE KINASE
Journal of Biological Chemistry, 1997Co-Authors: Roger Stäsche, Stephan Hinderlich, Karin Effertz, Lothar Lucka, Christoph Weise, Petra Moormann, Werner ReutterAbstract:N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a group of important molecules in biological recognition systems. Biosynthesis of Neu5Ac is initiated and regulated by its key enzyme, UDP-N-acetylglucosamine 2-Epimerase (UDP-GlcNAc 2-Epimerase, EC 5.1. 3.14)/N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S., Stasche, R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272, 24313-24318). In the present paper we report the isolation and characterization of a cDNA clone encoding this bifunctional enzyme. An open reading frame of 2166 base pairs encodes 722 amino acids with a predicted molecular mass of 79 kDa. The deduced amino acid sequence contains exact matches of the sequences of five peptides derived from tryptic cleavage of the enzyme. The recombinant bifunctional enzyme was expressed in COS7 cells, where it displayed both Epimerase and kinase activity. Distribution of UDP-GlcNAc 2-Epimerase/ManNAc kinase in the cytosol of several rat tissues was investigated by determining both specific enzyme activities. Secreting organs (liver, salivary glands, and intestinal mucosa) showed high specific activities of UDP-GlcNAc 2-Epimerase/ManNAc kinase, whereas significant levels of these activities were absent from other organs (lung, kidney, spleen, brain, heart, skeletal muscle, and testis). Northern blot analysis revealed no UDP-GlcNAc 2-Epimerase/ManNAc kinase mRNA in the non-secreting tissues.