Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase

Cid, Emili; Gomis, Roger R.; Geremia, R.; Guinovart, Joan J.; Ferrer, Juan C.

doi:10.1074/jbc.m005358200

Cited by 62 publications

(73 citation statements)

References 45 publications

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“…The deduced amino acid sequence closely matches the E-X 7 -E motif (Figure 2) that is found in eukaryotic glycosyltransferase family 3 proteins such as glycogen synthase (Cid et al, 2000(Cid et al, , 2002. However, similar motifs are found in four other families of retaining glycosyltransferases family members.…”

Section: Identification Of Candidate Genes In Starch and Floridoside supporting

confidence: 66%

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

et al. 2004

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When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56°C. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria, 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria.

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Section: Identification Of Candidate Genes In Starch and Floridoside supporting

confidence: 66%

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

et al. 2004

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“…The two glutamate residues of the EX 7 E motif have been proposed to be involved in catalysis as nucleophile but with mixed results. Whereas studies of the human muscle glycogen synthase (40) and AceA mannosyltransferase from Acetobacter xylinum (41) indicate that the first Glu residue is critical for enzyme activity, Gpi3, involved in glycosylphosphatidylinositol biosynthesis of S. cerevisiae, showed that the second Glu residue is of greater importance (42). As depicted in Fig.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

Kämpf

Absmanner

Schwarz

et al. 2009

Journal of Biological Chemistry

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N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the ␣1,3-and the ␣1,6-mannose-linked residue from GDP-mannose to Man 1 GlcNAc 2 -PP-Dol forming the Man 3 GlcNAc 2 -PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N-or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX 7 E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys 230 , as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.Asparagine-linked glycosylation is an essential protein modification highly conserved in eukaryotes (1-4), and several features of this pathway even occur in prokaryotes (5-7). In eukaryotes, biosynthesis of N-glycans starts with the assembly of the common core oligosaccharide precursor Glc 3 Man 9 GlcNAc 2 -PP-Dol, the glycan moiety of which is subsequently transferred onto selected Asn-Xaa-(Ser/Thr) acceptor sites of the nascent polypeptide chain by the oligosaccharyl-transferase complex (8 -10). The initial steps of the dolichol pathway up to Man 5 GlcNAc 2 -PP-Dol take place on the cytosolic site of the endoplasmic reticulum (ER), 2 using sugar nucleotides as glycosyl donors. Upon translocation of the heptasaccharide to the luminal site, which is facilitated by Rft1 (11) and another not yet identified protein (12), it is extended by four mannose and three glucose residues deriving from Man-P-Dol and Glc-P-Dol. It has been demonstrated that the pathway operates sequentially in an ordered fashion based on differences in the substrate specificity of the various glycosyltransferases (13). In the yeast Saccharomyces cerevisiae, alg mutants (for asparagine-linked glycosylation) have been isolated, defective in lipid-linked oligosaccharide (LLO) assembly (14 -17), and shown to be invaluable to define the pathway as well as to isolate the genes encoding the respective glycosyltransferases by complementing a particular phenotype characteristic of the respective mutant. Likewise various mutant cell lines from mammalian origin have been described that produce truncated lipid-linked oligosaccharides (18 -20).One of the tempe...

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“…2), is in a highly conserved stretch of residues (supplemental Fig. S1) that includes the putative active-site nucleophile Glu-509 (30). Mutations within site-4 were the least detrimental to catalysis using glycogen but the most detrimental to the enzyme's ability to utilize maltooctaose (Tables 2 and 3).…”

Section: Discussionmentioning

confidence: 99%

Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

Baskaran

Chikwana

Contreras

et al. 2011

Journal of Biological Chemistry

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Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V max /S 0.5 for glycogen by 40-and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V max /S 0.5 for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (⌬gsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.Glycogen synthase was the first reported intracellular target of insulin, and the enzyme catalyzes the linear polymerization of glucose residues from activated sugar donor molecules to the nonreducing end of the glycogen chain. Recent structural studies have shown that the enzyme folds into two Rossmann foldlike domains, with a deep cleft in between harboring the active site (1-3). Although the basic fold is conserved between the prokaryotic, archaeal, and eukaryotic enzymes, there are multiple sequence insertions in the eukaryotic enzymes. The largest of these insertions (a long coiled-coil insert in the C-terminal domain) gives rise to their unique tetrameric arrangement as well as the structural plasticity necessary for the complex regulation of glycogen synthase activity in eukaryotes (3). Furthermore, a conserved arginine cluster present in the C-terminal region of the eukaryotic enzymes mediates the sensitivity to inhibition by phosphorylation and activation by glucose 6-phosphate (4, 5). In our recent structural studies, we demonstrated that the middle two arginine residues 3 are necessary and sufficient to confer regulation by glucose 6-phosphate and that the first three arginine residues (Arg-580, Arg-581, and Arg-583) are required for full regulatory response to phosphorylation (3).The yeast Saccharomyces cerevisiae possesses two genes encoding glycogen synthase, GSY1 and GSY2, wh...

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Identification of Two Essential Glutamic Acid Residues in Glycogen Synthase

Cited by 62 publications

References 45 publications

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

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