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Andreas Blennow - One of the best experts on this subject based on the ideXlab platform.
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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, 2005Co-Authors: Lone Baunsgaard, René Mikkelsen, Henrik Lütken, Mikkel A. Glaring, Tam Thanh Pham, Andreas BlennowAbstract:An Arabidopsis thaliana gene encoding a homologue of the potato Alpha-Glucan, 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 Alpha-Glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.
The Biochemical journal, 2005Co-Authors: René Mikkelsen, Andreas BlennowAbstract:The potato tuber (Solanum tuberosum) GWD (Alpha-Glucan, 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 Alpha-Glucan,water dikinase, the starch phosphorylating enzyme.
The Biochemical journal, 2004Co-Authors: René Mikkelsen, Lone Baunsgaard, Andreas BlennowAbstract:GWD (Alpha-Glucan,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.
Marcello Iacomini - One of the best experts on this subject based on the ideXlab platform.
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A procedure for characterizing Glucans synthesized by purified enzymes of cariogenic Streptococcus mutans.
International journal of biological macromolecules, 2010Co-Authors: C.p. Aires, Hyun Koo, Guilherme L. Sassaki, Marcello Iacomini, Jaime Aparecido CuryAbstract: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 Alpha-Glucan contained 60% of (1-->6)-linkages and 17% of branching while insoluble Alpha-Glucan 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, 2005Co-Authors: Elaine R. Carbonero, Guilherme L. Sassaki, Anderson V. Montai, Caroline G. Mellinger, Sionara Eliasaro, Philip A.j. Gorin, Marcello IacominiAbstract: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)-Alpha-Glucan] 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, 2003Co-Authors: Lucimara M C Cordeiro, Philip A.j. Gorin, Elfriede Stockerworgotter, Marcello IacominiAbstract: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 Alpha-Glucan (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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effect of a soluble α d Glucan from the lichenized fungus ramalina celastri on macrophage activity
International Immunopharmacology, 2002Co-Authors: Patricia Ma Stuelpcampelo, Elaine R. Carbonero, Philip A.j. Gorin, Maria Benigna M Oliveira, Ana Maria Dos Anjos Carneiro Leao, Marcello IacominiAbstract:An Alpha-Glucan from the lichen Ramalina celastri has previously been demonstrated to have cytotoxic effects against HeLa cells. This polysaccharide was studied using Sarcoma-180 cells as tumoral model, and its effects on peritoneal exudate cells, namely, hydrogen peroxide production, phagocytic activity and cell eliciting activity are evaluated. Tumors developing in animals treated with the Glucan at a dose of 200 mg kg(-1), had a tumor size approximately 80% smaller than that of the control group, showing an impairment of tumor establishment. The polysaccharide was injected into mice not bearing a tumor and after 7, 15 and 30 days the cells were collected from the peritonea. The number of peritoneal cells increased approximately 130% 7 days after inoculation, and then gradually decreased. Hydrogen peroxide production was 75% greater 7 and 15 days after inoculation, on in vitro phorbol myristate acetate (PMA) triggering. Without PMA, the difference in hydrogen peroxide production was not significant. Phagocytic assays using fluorescent beads showed that the uptake increased 7 and 15 days after inoculation, when compared with the control. These results thus suggest a possible role of the R. celastri Glucan as a biological response modifier (BRM).
René Mikkelsen - One of the best experts on this subject based on the ideXlab platform.
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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, 2005Co-Authors: Lone Baunsgaard, René Mikkelsen, Henrik Lütken, Mikkel A. Glaring, Tam Thanh Pham, Andreas BlennowAbstract:An Arabidopsis thaliana gene encoding a homologue of the potato Alpha-Glucan, 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 Alpha-Glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.
The Biochemical journal, 2005Co-Authors: René Mikkelsen, Andreas BlennowAbstract:The potato tuber (Solanum tuberosum) GWD (Alpha-Glucan, 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 Alpha-Glucan,water dikinase, the starch phosphorylating enzyme.
The Biochemical journal, 2004Co-Authors: René Mikkelsen, Lone Baunsgaard, Andreas BlennowAbstract:GWD (Alpha-Glucan,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.
Stephen G. Withers - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and kinetic evaluation of 4-deoxymaltopentaose and 4-deoxymaltohexaose as inhibitors of muscle and potato Alpha-Glucan phosphorylases.
Biochemical Journal, 1999Co-Authors: Renee Mosi, Stephen G. WithersAbstract:Alpha-Glucan phosphorylases degrade linear or branched oligosaccharides via a glycosyl transfer reaction, occurring with retention of configuration, to generate Alpha-glucose-1-phosphate (G1P). We report here the chemoenzymic synthesis of two incompetent oligosaccharide substrate analogues, 4-deoxymaltohexaose (4DG6) and 4-deoxymaltopentaose (4DG5), for use in probing this mechanism. A kinetic analysis of the interactions of 4DG5 and 4DG6 with both muscle and potato phosphorylases was completed to provide insight into the nature of the binding mode of oligosaccharide to phosphorylase. The 4-deoxy-oligosaccharides bind competitively with maltopentaose and non-competitively with respect to orthophosphate or G1P in each case, indicating binding in the oligosaccharide binding site. Further, 4DG5 and 4DG6 were found to bind to potato and muscle phosphorylases some 10-40-fold tighter than does maltopentaose. Similar increases in affinity as a consequence of 4-deoxygenation were observed previously for the binding of polymeric glycogen analogues to rabbit muscle phosphorylase [Withers (1990) Carbohydr. Res. 196, 61-73].
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Measurement of active-site homology between potato and rabbit muscle Alpha-Glucan phosphorylases through use of a linear free energy relationship.
Biochemistry, 1990Co-Authors: Stephen G. Withers, Karen RupitzAbstract:The Michaelis-Menten parameters (Vmax and Km) for turnover of an extensive series of deoxy and deoxyfluoro derivatives of Alpha-D-glucopyranosyl phosphate by the Alpha-Glucan phosphorylase from potato tuber have been determined. Very large rate reductions are observed as a consequence of each substitution, primarily due to losses in specific binding interactions, most likely hydrogen bonding, at the enzymic transition state. Comparison of the Vmax/Km values so determined with those measured for rabbit muscle Alpha-Glucan phosphorylase [Street et al. (1989) Biochemistry 28, 1581] reveals an astonishingly similar specificity, especially in light of the phylogenetic separation of their host organisms. This indicates that very similar hydrogen-bonding interactions between the enzyme and the substrate must be present at the transition states for the two enzymic reactions; therefore, they have very similar active sites. Quantitation of this similarity is achieved by plotting the logarithm of the Vmax/Km value for each substrate analogue with the potato enzyme against the same parameter for the muscle enzyme, yielding straight lines (p = 0.998 and 0.999) of slope 1.0 and 1.2 for the deoxy and deoxyfluoro substrates, respectively. Since the correlation coefficient of such plots is a direct measure of the similarity of the two transition-state complexes, thus of the enzyme active sites, it can be used as a measure of active-site homology between the two enzymes. The extremely high homology observed in this case is consistent with the observed sequence homology at the active site.
Frans M Klis - One of the best experts on this subject based on the ideXlab platform.
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Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans.
Fungal genetics and biology : FG & B, 2009Co-Authors: Piet W J De Groot, Bernd W Brandt, Hiroyuki Horiuchi, Arthur F J Ram, Chris G De Koster, Frans M KlisAbstract:Knowledge of the mechanisms underlying cell wall biosynthesis in Aspergillus spp. is of high relevance to medicine and food safety, and for biotechnological applications. The cell wall of Aspergillus nidulans is composed of galactomannoproteins, 1,3-Alpha-Glucan, beta-Glucans, and chitin. Here, we present a comprehensive inventory of the cell wall-related genes in A. nidulans. This includes glycan-synthetic and glycan-processing enzymes, spore wall maturation enzymes, GPI-anchor processing enzymes, GPI proteins and hydrophobins, and signaling proteins of the cell wall integrity pathway. All major known fungal cell wall-related genes are represented, except for Pir-CWPs. Importantly, we have identified a gene product that is possibly involved in the synthesis of 1,3/1,4-beta-Glucan, and we propose that four predicted GPI proteins, a mixed-linked beta-Glucanase and three amylase-like Alpha-Glucanases, may have transglucosidic activities pertaining to the processing of 1,3/1,4-beta-Glucan and 1,3/1,4-Alpha-Glucan, respectively. We further present an updated survey of putative GPI proteins. Finally, we present mass spectrometric evidence suggesting the presence of at least twelve covalently-linked cell wall proteins in the hyphal wall of A. nidulans, including ten predicted GPI proteins, most of which belong to carbohydrate-active enzyme families that are also found in the walls of ascomycetous yeasts.
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the structure of cell wall Alpha Glucan from fission yeast
Glycobiology, 2005Co-Authors: Christian H Grun, Frans Hochstenbach, Bruno M Humbel, A J Verkleij, J H Sietsma, Frans M Klis, Johannis P Kamerling, Johannes F G VliegenthartAbstract:Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1rarr3)-beta-Glucan, have been studied extensively, whereas little is known about Alpha-Glucan. Here we describe the chemical structure of Alpha-Glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type Alpha-Glucan was found to consist of a single population of linear glucose polymers, sim260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of sim120 (1rarr3)-linked Alpha-d-glucose residues and some (1rarr4)-linked Alpha-d-glucose residues at the reducing end. By contrast, Alpha-Glucan of an Alpha-Glucan synthase mutant with an aberrant cell morphology and reduced Alpha-Glucan levels consisted of a single chain only. We propose that Alpha-Glucan biosynthesis involves an ordered series of events, whereby two Alpha-Glucan chains are coupled to create mature cell wall Alpha-Glucan. This mature form of cell wall Alpha-Glucan is essential for fission-yeast morphogenesis.
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Identification of a putative Alpha-Glucan synthase essential for cell wall construction and morphogenesis in fission yeast
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Frans Hochstenbach, Frans M Klis, Herman Van Den Ende, Elly Van Donselaar, Peter J. Peters, Richard D. KlausnerAbstract:The cell wall protects fungi against lysis and determines their cell shape. Alpha-Glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1+, which encodes a putative Alpha-Glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for Alpha-Glucan assembly, namely an intracellular domain for Alpha-Glucan synthesis and an extracellular domain speculated to cross-link or remodel Alpha-Glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of Alpha-Glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall Alpha-Glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that Alpha-Glucan is essential for fission yeast morphogenesis.