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Andreas Blennow – One of the best experts on this subject based on the ideXlab platform.

  • A novel isoform of Glucan, water dikinase phosphorylates pre‐phosphorylated α‐Glucans and is involved in starch degradation in Arabidopsis
    The Plant journal : for cell and molecular biology, 2005
    Co-Authors: Lone Baunsgaard, René Mikkelsen, Henrik Lütken, Mikkel A. Glaring, Tam Thanh Pham, Andreas Blennow

    Abstract:

    An Arabidopsis thaliana gene encoding a homologue of the potato AlphaGlucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.

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  • Functional domain organization of the potato AlphaGlucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.
    The Biochemical journal, 2005
    Co-Authors: René Mikkelsen, Andreas Blennow

    Abstract:

    The potato tuber (Solanum tuberosum) GWD (AlphaGlucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure-function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the Glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and Glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP–>E-P+AMP+P(i) (where P(i) stands for orthophosphate) partial reaction, but not the E-P+Glucan–>E+Glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

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  • Functional characterization of AlphaGlucan,water dikinase, the starch phosphorylating enzyme.
    The Biochemical journal, 2004
    Co-Authors: René Mikkelsen, Lone Baunsgaard, Andreas Blennow

    Abstract:

    GWD (AlphaGlucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of Alpha-1,6 branch points in its polyGlucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.

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Marcello Iacomini – One of the best experts on this subject based on the ideXlab platform.

  • A procedure for characterizing Glucans synthesized by purified enzymes of cariogenic Streptococcus mutans.
    International journal of biological macromolecules, 2010
    Co-Authors: C.p. Aires, Hyun Koo, Guilherme L. Sassaki, Marcello Iacomini, Jaime Aparecido Cury

    Abstract:

    Extracellular polysaccharide is a virulence factor of the most cariogenic bacteria, Streptococcus mutans. We describe a procedure for chemical analysis of this polysaccharide of S. mutans, using a well-known Glucan synthesized by purified enzymes of cariogenic streptococci. Soluble and insoluble Glucan from glucosyltransferase D and B respectively, were prepared and analyzed by nuclear magnetic resonance spectroscopy and methylation. Soluble AlphaGlucan contained 60% of (1–>6)-linkages and 17% of branching while insoluble AlphaGlucan was composed of 57% of (1–>3)- and 28% of (1–>6)-linkages with 8% branching. The described procedure could be important focusing future studies on in vivo biofilm.

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  • Glucans of lichenized fungi: significance for taxonomy of the genera Parmotrema and Rimelia
    Phytochemistry, 2005
    Co-Authors: Elaine R. Carbonero, Guilherme L. Sassaki, Anderson V. Montai, Caroline G. Mellinger, Sionara Eliasaro, Philip A.j. Gorin, Marcello Iacomini

    Abstract:

    The Glucans of lichenized fungi are an important class of polysaccharides with structural and chemotaxonomic roles. The water-insoluble Glucans of the genus Parmotrema (P. austrosinense, P. delicatulum, P. mantiqueirense, P. schindleri, and P. tinctorum) and those of Rimelia (R. cetrata and R. reticulata), were investigated in order to evaluate the significance in chemotyping, with nigeran [(1–>3),(1–>4)-AlphaGlucan] and lichenan [(1–>3),(1–>4)-beta-Glucan] characterized using (1)H and (13)C NMR, methylation analysis, and controlled Smith degradations. Results from all species were similar, suggesting that Glucan chemistry does not support separation of Rimelia from Parmotrema.

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  • comparative studies of the polysaccharides from species of the genus ramalina lichenized fungi of three distinct habitats
    Phytochemistry, 2003
    Co-Authors: Lucimara M C Cordeiro, Philip A.j. Gorin, Elfriede Stockerworgotter, Marcello Iacomini

    Abstract:

    Several structurally different Glucans (Alpha– and beta-) and galactomannans were characterized as components of four species of the genus Ramalina, namely R. dendriscoides, R. fraxinea, R. gracilis and R. peruviana. Freeze-thawing treatment of hot aqueous extracts furnished as precipitates (PW) linear Alpha-D-Glucans of the nigeran type, with regularly distributed (1–>3)- and (1–>4)-linkages in a 1:1 ratio. The supernatants (SW) contained Alpha-D-Glucans with (1–>3)- and (1–>4)-linkages in a molar ratio of 3:1. The lichen residues were then extracted with 2% aq. KOH, and the resulting extracts submitted to the freeze-thawing treatment, giving rise to precipitates (PK2) of a mixture of AlphaGlucan (nigeran) and beta-Glucan, which were suspended in aqueous 0.5% NaOH at 50 degrees C, dissolving preferentially the beta-Glucan. These were linear with (1–>3)-linkages (laminaran). The mother liquor of the KOH extractions (2% and 10% aq. KOH) was treated with Fehling’s solution to give precipitates (galactomannans). The galactomannans are related, having (1–>6)-linked Alpha-D-mannopyranosyl main chains, substituted at O-4 and in a small proportion at O-2,4 by beta-D-galactopyranosyl units. Despite the different habitats of these lichenized fungi, all species studied in this investigation have a similar pool of polysaccharides.

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René Mikkelsen – One of the best experts on this subject based on the ideXlab platform.

  • A novel isoform of Glucan, water dikinase phosphorylates pre‐phosphorylated α‐Glucans and is involved in starch degradation in Arabidopsis
    The Plant journal : for cell and molecular biology, 2005
    Co-Authors: Lone Baunsgaard, René Mikkelsen, Henrik Lütken, Mikkel A. Glaring, Tam Thanh Pham, Andreas Blennow

    Abstract:

    An Arabidopsis thaliana gene encoding a homologue of the potato AlphaGlucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.

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  • Functional domain organization of the potato AlphaGlucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.
    The Biochemical journal, 2005
    Co-Authors: René Mikkelsen, Andreas Blennow

    Abstract:

    The potato tuber (Solanum tuberosum) GWD (AlphaGlucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure-function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the Glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and Glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP–>E-P+AMP+P(i) (where P(i) stands for orthophosphate) partial reaction, but not the E-P+Glucan–>E+Glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

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  • Functional characterization of AlphaGlucan,water dikinase, the starch phosphorylating enzyme.
    The Biochemical journal, 2004
    Co-Authors: René Mikkelsen, Lone Baunsgaard, Andreas Blennow

    Abstract:

    GWD (AlphaGlucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of Alpha-1,6 branch points in its polyGlucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.

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