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Stephan Hinderlich – One of the best experts on this subject based on the ideXlab platform.

  • UDP-GlcNAc 2-Epimerase/ManNAc Kinase (GNE): A Master Regulator of Sialic Acid Synthesis.
    Topics in Current Chemistry, 2013
    Co-Authors: Stephan Hinderlich, Tal Yardeni, Rüdiger Horstkorte, Wenke Weidemann, Marjan Huizing

    Abstract:

    UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase is the key enzyme of sialic acid biosynthesis in vertebrates. It catalyzes the first two steps of the cytosolic formation of CMP-N-acetylneuraminic acid from UDP-N-acetylglucosamine. In this review we give an overview of structure, biochemistry, and genetics of the bifunctional enzyme and its complex regulation. Furthermore, we will focus on diseases related to UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase.

  • Biochemical characterization of human and murine isoforms of UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase (GNE)
    Glycoconjugate Journal, 2009
    Co-Authors: Stefan O. Reinke, Colin Eidenschink, Stephan Hinderlich

    Abstract:

    The bifunctional enzyme UDP- N -acetylglucosamine 2-Epimerase/ N -acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-Epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli , only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.

  • Influence of UDP-GlcNAc 2-Epimerase/ManNAc kinase mutant proteins on hereditary inclusion body myopathy
    BIOCHEMISTRY-US, 2006
    Co-Authors: Stephan Hinderlich

    Abstract:

    Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutations in UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported to cause the disease. Ten of them, representing mutations in both functional domains of GNE, were recombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reduction in the two known GNE activities, thus revealing that mutations may also influence the function of the domain not harboring them. The extent of reduction strongly differs among the point mutants, ranging from only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activity in D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined by structural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis, respectively. We therefore generated models of the three-dimensional structures of the epimerase and the kinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-Epimerase and glucokinase, respectively, and determined the localization of the HIBM mutations within these proteins. Whereas in the kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerase domain are mostly located at the protein surface. Otherwise, the different mutations result in different enzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role of the enzymatic function of GNE in the disease mechanism.

Werner Reutter – One of the best experts on this subject based on the ideXlab platform.

  • Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase.
    Biochemical Journal, 2004
    Co-Authors: Astrid Blume, Werner Reutter, Rüdiger Horstkorte, Lothar Lucka, Wenke Weidemann, Ulrich Stelzl, Erich E. Wanker, Peter Donner, Stephan Hinderlich

    Abstract:

    UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase is a bifunctional enzyme, which initiates and regulates sialic acid biosynthesis. Sialic acids are important compounds of mammalian glycoconjugates, mediating several biological processes, such as cell-cell or cell-matrix interactions. In order to characterize the function of UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase, a number of deletion mutants were generated, lacking either parts of the N-terminal epimerase or the C-terminal kinase domain. N-terminal deletion of only 39 amino acids results in a complete loss of epimerase activity. Deletions in the C-terminal part result in a reduction or complete loss of kinase activity, depending on the size of the deletion. Deletions at either the N- or the C-terminus also result in a reduction of the other enzyme activity. These results indicate that a separate expression of both domains is possible, but that a strong intramolecular dependency of the two domains has arisen during evolution of the enzyme. N-terminal, as well as C-terminal, mutants tend to form trimers, in addition to the hexameric structure of the native enzyme. These results and yeast two-hybrid experiments show that structures required for dimerization are localized within the kinase domain, and a potential trimerization site is possibly located in a region between the two domains. In conclusion, our results reveal that the activities, as well as the oligomeric structure, of this bifunctional enzyme seem to be organized and regulated in a complex manner.

  • 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.
    FEBS Letters, 2004
    Co-Authors: Stephan Hinderlich, Rüdiger Horstkorte, Kevin J. Yarema, Lars R. Mantey, Ilan Salama, Iris Eisenberg, Tamara Potikha, Zohar Argov, Menachem Sadeh, Werner Reutter

    Abstract:

    Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-Epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.

  • UDP-N-acetylglucosamine 2-Epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells.
    Protein Expression and Purification, 2004
    Co-Authors: Astrid Blume, Stephan Hinderlich, Werner Reutter, Peter Donner, Darius Ghaderi, Viola Liebich, Lothar Lucka

    Abstract:

    Abstract UDP-GlcNAc 2-Epimerase/ManNAc kinase is the key enzyme of sialic acid biosynthesis in mammals. Its functional expression is a prerequisite for early embryogenesis and for the synthesis of several cell recognition motifs in adult organism. This bifunctional enzyme is involved in the development of different diseases like sialuria or hereditary inclusion body myopathy. For a detailed understanding of the enzyme, large amounts of the pure active protein are needed. Different heterologous cell systems were therefore analyzed for the enzyme, which was found to be functionally expressed in Escherichia coli , the yeast strains Saccharomyces cerevisiae and Pichia pastoris , and insect cells. In all these cell types, the expressed enzyme displayed both epimerase and kinase activities. In E. coli , up to 2 mg protein/l cell culture was expressed, in yeast cells only 0.4 mg/L, while up to 100 mg/L, were detected in insect cells. In all three cell systems, insoluble protein aggregates were also observed. Purification from E. coli resulted in 100 μg/L pure and structurally intact protein. For insect cells, purification methods were established which resulted in up to 50 mg/L pure, soluble, and active protein. In summary, expression and purification of the UDP-GlcNAc 2-Epimerase/ManNAc kinase in Sf-900 cells can yield the milligram amounts of protein required for structural characterization of the enzyme. However, the easier expression in E. coli and yeast provides sufficient quantities for enzymatic and kinetic characterization.

Svein Valla – One of the best experts on this subject based on the ideXlab platform.

  • structural and mutational characterization of the catalytic a module of the mannuronan c 5 epimerase alge4 from azotobacter vinelandii
    Journal of Biological Chemistry, 2008
    Co-Authors: Henriette J Rozeboom, Svein Valla, Helga Ertesvag, Tonje M Bjerkan, Kor H Kalk, Synnove Holtan, Finn Lillelund Aachmann, Bauke W Dijkstra

    Abstract:

    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.

  • the catalytic activities of the bifunctional azotobacter vinelandii mannuronan c 5 epimerase and alginate lyase alge7 probably originate from the same active site in the enzyme
    Journal of Biological Chemistry, 2001
    Co-Authors: Britt Iren Glaerum Svanem, Wenche Strand, Helga Ertesvag, Gudmund Skjakbraek, Martin Hartmann, Tristan Barbeyron, Svein Valla

    Abstract:

    Abstract The Azotobacter vinelandii genome encodes a family of seven secreted Ca2+-dependent epimerases (AlgE1–7) catalyzing the polymer level epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in the commercially important polysaccharide alginate. AlgE1–7 are composed of two types of protein modules, A and R, and the A-modules have previously been found to be sufficient for epimerization. AlgE7 is both an epimerase and an alginase, and here we show that the lyase activity is Ca2+-dependent and also responds similarly to the epimerases in the presence of other divalent cations. The AlgE7 lyase degraded M-rich alginates and a relatively G-rich alginate from the brown algae Macrocystis pyrifera most effectively, producing oligomers of 4 (mannuronan) to 7 units. The sequences cleaved were mainly G↓MM and/or G↓GM. Since G-moieties dominated at the reducing ends even when mannuronan was used as substrate, the AlgE7 epimerase probably stimulates the lyase pathway, indicating a complex interplay between the two activities. A truncated form of AlgE1 (AlgE1-1) was converted to a combined epimerase and lyase by replacing the 5′-798 base pairs in the algE1-1 gene with the corresponding A-module-encoding DNA sequence from algE7. Furthermore, substitution of an aspartic acid residue at position 152 with glycine in AlgE7A eliminated almost all of both the lyase and epimerase activities. Epimerization and lyase activity are believed to be mechanistically related, and the results reported here strongly support this hypothesis by suggesting that the same enzymatic site can catalyze both reactions.

  • cloning and expression of an azotobacter vinelandii mannuronan c 5 epimerase gene
    Journal of Bacteriology, 1994
    Co-Authors: Helga Ertesvag, Gudmund Skjakbraek, Berit Doseth, Bjorn Larsen, Svein Valla

    Abstract:

    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.