2-Epimerase

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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.

  • 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.

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.

  • 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.

  • 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.

  • C‐Glycosidic UDP‐GlcNAc Analogues as Inhibitors of UDP‐GlcNAc 2‐Epimerase
    European Journal of Organic Chemistry, 2004
    Co-Authors: Florian Stolz, Stephan Hinderlich, Werner Reutter, Astrid Blume, Richard R. Schmidt
    Abstract:

    The first step in the biosynthesis of neuraminic acid, the “epimerisation” of UDP-GlcNAc to ManNAc, is catalyzed by UDP-GlcNAc 2-Epimerase. In this paper we report the synthesis of the C-glycosidic UDP-GlcNAc analogues 1−5 as substrate-based inhibitors of this enzyme. The focus is on the optimal distance and geometry of the connection between the sugar and the UDP-moiety, which are both important for recognition by UDP-GlcNAc 2-Epimerase. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

  • Epigenetically mediated loss of UDP-GlcNAc 2-Epimerase/ManNAc kinase expression in hyposialylated cell lines.
    Biochemical and Biophysical Research Communications, 2003
    Co-Authors: Cornelia Oetke, Stephan Hinderlich, Werner Reutter, Michael Pawlita
    Abstract:

    The bifunctional enzyme UDP-GlcNAc 2-Epimerase/ManNAc kinase is the key enzyme in sialic acid biosynthesis. Loss of UDP-GlcNAc 2-Epimerase activity results in a hyposialylated phenotype as shown for two human hematopoietic cell lines that lack the specific mRNA. We found that treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine (5-aza-dC) restored the UDP-GlcNAc 2-Epimerase/ManNAc kinase mRNA, as well as enzyme activity and cell surface sialylation. Increase of UDP-GlcNAc 2-Epimerase activity by 5-aza-dC treatment was also found for a rat Morris hepatoma cell line. These results indicate a regulation of UDP-GlcNAc 2-Epimerase/ManNAc kinase expression on the transcriptional level by DNA methylation.

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, Helga Ertesvag, Svein Valla, 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.

Marjan Huizing - 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.

  • identification tissue distribution and molecular modeling of novel human isoforms of the key enzyme in sialic acid synthesis udp glcnac 2 epimerase mannac kinase
    Biochemistry, 2011
    Co-Authors: Tal Yardeni, William A. Gahl, Carla Ciccone, Tsering Choekyi, Katherine Jacobs, Katherine Patzel, Yair Anikster, Natalya Kurochkina, Marjan Huizing
    Abstract:

    The bifunctional enzyme uridine diphosphate (UDP)1-N-acetylglucosamine (GlcNAc) 2- epimerase/N-acetylmannosamine (ManNAc) kinase (GNE), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (Neu5Ac) (1, 2). Neu5Ac is the most abundant mammalian sialic acid and the precursor of most naturally existing sialic acids (3). Sialic acids are negatively charged, terminal residues on glycoconjugates, and assist in many cellular functions including cell-cell interactions, proliferation, and viral or bacterial infections (3, 4). The GNE enzyme consists of two enzymatic domains. The N-terminal domain carries out UDP-GlcNAc epimerase function, whereas the Cterminal domain is responsible for ManNAc kinase activity. In mammals, the end product of sialic acid synthesis, CMP-Neu5Ac, feedback-inhibits UDP-GlcNAc 2-Epimerase activity of GNE by binding to its allosteric site (5). Two distinct human disorders, sialuria (OMIM 269921) and hereditary inclusion body myopathy (HIBM; OMIM 600737), are associated with predominantly missense mutations in GNE. Sialuria is an autosomal dominant disorder characterized by coarse facies, variable developmental delay, hepatomegaly and recurrent infections. To date, only seven sialuria patients are described worldwide. All patients have a heterozygous missense mutation affecting the allosteric site of GNE, leading to loss of feedback-inhibition of GNE-epimerase activity by CMP-Neu5Ac, resulting in excessive sialic acid production (6, 7). HIBM and its allelic Japanese disorder, distal myopathy with rimmed vacuoles, or DMRV (OMIM 605820), is an autosomal recessive neuromuscular disorder of adult onset, characterized by slowly progressive muscle weakness and atrophy. More than 500 HIBM\DMRV patients exist worldwide, harboring over 60 GNE mutations. HIBM\DMRV patients have recessive (predominantly missense) mutations in either enzymatic domain of GNE, leading to decreased enzyme activity and, presumably, decreased sialic acid production (2, 8, 9). Whether hyposialylation is the main cause of the neuromuscular symptoms in HIBM\DMRV patients remains unknown. In prokaryotes, GNE epimerase and kinase functions are carried out by two separate enzymes, and prokaryotic 2-Epimerases have no allosteric feedback inhibition. In mammals, a bifunctional enzyme might have evolved by gene fusion of the two independent enzymes responsible for epimerase/kinase activity. Similarities between mammalian GNE N-terminal regions with prokaryotic UDP-GlcNAc 2-Epimerases and mammalian GNE C-terminal regions with members of the sugar kinase superfamily previously assisted in identifying characteristic motifs of the GNE epimerase and kinase enzymatic domains (10, 11). Bacterial 2-Epimerases are allosterically regulated by its substrate UDP-GlcNAc. A structural basis for allosteric activation was demonstrated by a crystallographic analysis of the B. subtilis 2 2-Epimerase in complex with the reaction intermediate UDP (12). In addition, the crystallographic structures of the E. coli GNE enzyme unbound and in complex with UDP-N-acetylglucosamine (pdb code 1f6d, 1vgv), and the V. cholera (pdb code 1dzc) and B. anthracis (pdb code 3beo) enzymes in complex with UDP-N-acetylglucosamine were solved. Similarity of the N-terminal domain of the H. sapiens GNE to V. cholera (27% homology), E. coli (20% homology) and B. anthracis (18% homology) 2-Epimerases was used to model its three-dimensional structure. In previous studies, structural elements and important ATP, ADP, Mg2+ and substrate-binding amino acids were assigned on the basis of these similarities (10, 11). The N-terminal epimerase domain of the human GNE enzyme contains two α/β domains (domains I and II) that form a cleft at the domain interface harboring the active site. Topology of both domains is similar to the Rossmann dinucleotide binding fold (13). Rossmann fold domains are conserved among mammalian and bacterial 2- epimerases. The human N-terminal GNE epimerase domain has a 7-stranded parallel β-sheet sandwiched between a total of 7 α-helices. The C-terminus of the GNE epimerase domain contains a 6-stranded β-sheet surrounded by a total of 7 α-helices (11). Other carriers of the Rossmann fold, including glycosyltransferases and the epimerase domains of 2-Epimerases, have similar N-terminal and C-terminal domains (14, 15). The crystallographic structure of the ManNAc kinase domain of human GNE is solved at 2.84 A resolution (pdb code 3eo3) (16). Residues 409–431 of the mammalian GNE ManNAc kinase domain showed similarities with the phosphate 1 motif of the ATP-binding domain of eukaryotic hexokinases (10). Similar to hexokinases (17), mammalian GNE ManNAc kinase contains a 5-stranded β-sheet β3β2β1β4β5 with β2 being anti-parallel to four other parallel strands with a pair of parallel alpha-helices located on each side of the β-sheet (Domain I). Another 5-stranded β-sheet β8β7β6β9β1 with β7 being anti-parallel to four other parallel strands is surrounded by a pair of parallel α-helices on each side (Domain II). The structure of two similar domains involved in ATP binding is a common feature of the ASKHA (Acetate and Sugar Kinase/Hsp70/Actin) superfamily, described in detail for the bacterial poly(P)/ATP-glucomannokinase (18, 19). Recently, different human GNE mRNA splice variants and three predicted translated proteins, hGNE1, hGNE2 and hGNE3 were described (20, 21). Subsequently, two different mouse Gne mRNA splice variants were described, Gne1 and Gne2, together with their expression in selected tissues (22). In the current study we identified additional human isoforms hGNE4-8, and demonstrate expression of hGNE isoform transcripts in a wide variety of tissues. It is unknown which role these isoforms play in GNE regulation, or GNE-related disease pathology. Based on our previous modeling results of the hGNE1 isoform (11), we now analyze and compare the structural features, with respect to catalytic activity, ligand binding and allosteric regulation, of all eight human GNE isoforms.

Susumu Ito - One of the best experts on this subject based on the ideXlab platform.

  • Identification and distribution of cellobiose 2-Epimerase genes by a PCR-based metagenomic approach.
    Applied Microbiology and Biotechnology, 2014
    Co-Authors: Jun Wasaki, Hidenori Taguchi, Jun Watanabe, Takeshi Senoura, Hiroshi Akasaka, Kazuki Kawaguchi, Yosuke Komata, Kiyotoshi Hanashiro, Susumu Ito
    Abstract:

    Cellobiose 2-Epimerase (CE) catalyzes the reversible epimerization of cellobiose to 4-O-β-d-glucopyranosyl-d-mannose. By using a PCR-based metagenomic approach, 71 ce-like gene fragments were obtained from wide-ranging environmental samples such as sheep rumen, soils, sugar beet extracts, and anaerobic sewage sludge. The frequency of isolation of the fragments similar to known sequences varied depending on the nature of the samples used. The ce-like genes appeared to be widely distributed in environmental bacteria belonging to the phyla Bacteroidetes, Chloroflexi, Dictyoglomi, Firmicutes, Proteobacteria, Spirochaetes, and Verrucomicrobia. The phylogenetic analysis suggested that the cluster of CE and CE-like proteins was functionally and evolutionarily separated from that of N-acetyl-d-glucosamine 2-Epimerase (AGE) and AGE-like proteins. Two ce-like genes containing full-length ORFs, designated md1 and md2, were obtained by PCR and expressed in Escherichia coli. The recombinant mD1 and mD2 exhibited low K m values and high catalytic efficiencies (k cat/K m) for mannobiose compared with cellobiose, suggesting that they should be named mannobiose 2-Epimerase, which is involved in a new mannan catabolic pathway we proposed.

  • Features and applications of microbial sugar epimerases
    Applied Microbiology and Biotechnology, 2009
    Co-Authors: Susumu Ito
    Abstract:

    Sugar (carbohydrate) epimerases catalyze the reversible conversion of a sugar epimer into its counterpart form. More than 20 types of sugar epimerase have been reported to date, and their biological properties, catalytic mechanisms, and 3D structures are very diverse among them. Recently, microbial sugar epimerases have been characterized in detail. This review surveys the catalytic aspects of microbial epimerases, which are relevant for production of bioactive mono- and oligosaccharides.

  • Site-directed mutagenesis of possible catalytic residues of cellobiose 2-Epimerase from Ruminococcus albus.
    Biotechnology Letters, 2009
    Co-Authors: Shigeaki Ito, Shigeki Hamada, Hiroyuki Ito, Hirokazu Matsui, Tadahiro Ozawa, Hidenori Taguchi, Susumu Ito
    Abstract:

    The cellobiose 2-Epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-d-galactopyranosyl-d-mannose (epilactose). Based on the sequence alignment with N-acetyl-d-glucosamine 2-Epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)6 core barrel structure.

  • Identification of the cellobiose 2-Epimerase gene in the genome of Bacteroides fragilis NCTC 9343.
    Bioscience Biotechnology and Biochemistry, 2009
    Co-Authors: Takeshi Senoura, Shigeaki Ito, Shigeki Hamada, Hirokazu Matsui, Hidenori Taguchi, Jun Watanabe, Jun Wasaki, Satoru Fukiya, Atsushi Yokota, Susumu Ito
    Abstract:

    Cellobiose 2-Epimerase (CE, EC 5.1.3.11) catalyzes the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose. In this study, we found a CE gene in the genome sequence of non-cellulolytic Bacteroides fragilis NCTC 9343. The recombinant enzyme, expressed in Escherichia coli cells, catalyzed a hydroxyl stereoisomerism at the C-2 positions of the reducing terminal glucose and at the mannose moiety of cello-oligosaccharides, lactose, beta-mannobiose (4-O-beta-D-mannopyranosyl-D-mannose), and globotriose [O-alpha-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D-glucose]. The CE from B. fragilis showed less than 40% identity to reported functional CEs. It exhibited 44-63% identities to N-acyl-D-glucosamine 2-Epimerase-like hypothetical proteins of unknown function in bacterial genome sequences of the phyla Firmicutes, Bacteroidetes, Proteobacteria, Chloroflexi, and Verrucomicrobia. On the other hand, it showed less than 26% identity to functional N-acyl-D-glucosamine 2-Epimerases. Based on the amino acid homology and phylogenetic positions of the functional epimerases, we emphasize that many genes for putative N-acyl-D-glucosamine 2-Epimerases and related hypothetical proteins of unknown function reported to date in the bacterial genomes should be annotated as CE-like proteins or putative CEs.

  • cloning and sequencing of the gene for cellobiose 2 epimerase from a ruminal strain of eubacterium cellulosolvens
    Fems Microbiology Letters, 2008
    Co-Authors: Hidenori Taguchi, Shigeki Hamada, Hirokazu Matsui, Jun Watanabe, Jun Wasaki, Takeshi Senoura, Yasuo Kobayashi, Susumu Ito
    Abstract:

    Cellobiose 2-Epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-D-glucopyranosyl-D-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-D-glucosamine 2-Epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-D-galactopyranosyl-D-mannose).