<i>De novo</i>
            synthesis of bacterial glycogen:
            <i>Agrobacterium tumefaciens</i>
            glycogen synthase is involved in glucan initiation and elongation

Ugalde, Juan Esteban; Parodi, Armando J.; Ugalde, Rodolfo A.

doi:10.1073/pnas.1534787100

Cited by 84 publications

(84 citation statements)

References 20 publications

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“…6). The dual function of the glycogen synthase recently proposed for Agrobacterium tumefaciens [18] is consistent with this assumption. Furthermore, it concurs with some enzymatic measurements performed with glycogen accumulating E. coli cells.…”

Section: Discussionsupporting

confidence: 78%

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Albrecht¹,

Haebel

Koch³

et al. 2004

European Journal of Biochemistry

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Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His 6 ) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono-and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogenrelated enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.

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Section: Discussionsupporting

confidence: 78%

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Albrecht¹,

Haebel

Koch³

et al. 2004

European Journal of Biochemistry

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“…The second involves priming with oligomers of the substrate unit and is exemplified by maltooligosaccharides ((glucose) n , n ) 2-7 residues) in starch biosynthesis (11). The third involves self-priming by polymerase, a process exemplified by the bacterial glycogen synthase from Agrobacterium tumefaciens (12).…”

mentioning

confidence: 99%

Class III Polyhydroxybutyrate Synthase: Involvement in Chain Termination and Reinitiation

2005

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Polyhydroxybutyrate (PHB) synthase catalyzes the polymerization of (R)-3-hydroxybutyrylCoA (CoA ) coenzyme A) into high molecular weight PHB. Recombinant wild-type (wt) class III synthase from Allochromatium Vinosum (PhaCPhaE Av ), antibodies to this synthase and to PHB, and [ 14 C]-hydroxybutyryl-CoA (HB-CoA) have been used to detect oligomeric hydroxybutyrate (HB) units covalently bound to the synthase using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Although a distribution of products is typically observed, short (HB) n -bound synthases (designated species I) are most prevalent at low substrate to enzyme (S/E) ratios. Species I is similar to (HB) n -PhaC Av (n ) 3-10 at minimum) recently identified using D302A-PhaCPhaE Av (Tian, J., Sinskey, A. J., and Stubbe, J. (2005) Biochemistry 44, 1495-1503). Species I is shown to be an intermediate in the elongation process of PHB synthesis in vitro. The reaction catalyzed by the wt synthase in vitro was further studied under two sets of conditions: at high (70000) and low (<200) S/E ratios. At high S/E ratios, kinetic analysis of the reaction of HB-CoA with the wt synthase monitored using antibodies to PhaCPhaE Av and Western blotting revealed the disappearance of PhaC Av at early time points and its reappearance as the molecular weight of the PHB approached 1.8 MDa. At low S/E ratios, species I was observed to increase with time after complete consumption of all of the HB-CoA. The results from studies under both sets of conditions suggest that an inherent property of the synthase is chain termination and reinitiation.Polyhydroxyalkanoate (PHA 1 ) synthases catalyze the polymerization of (R)-3-hydroxyalkanoate-CoA into high molecular weight polyoxoesters under nutrient-limited conditions in the presence of a carbon source (1-4). PHA synthases are representative of enzymes involved in nontemplate-driven polymerization processes in which a watersoluble substrate is transformed into a water-insoluble granule or inclusion without a genetically determined template. The thermoplastic or elastomeric properties of PHAs (depending on the substrate) have provided the impetus for many research groups to study the mechanism and the regulation of the polymerization process, as well as the substrate specificity of the synthase. The long-range goal is to make materials with novel properties in an economically competitive fashion with oil-based polymers (5).The classification of PHA synthases, based on substrate specificity and subunit composition, has been described in detail in several reviews (6, 7). Our laboratory has focused on the class I and class III synthases that use (R)-3-hydroxybutyryl-CoA (HB-CoA) to form polyhydroxybutyrate (PHB). The class I synthase from the prototypical Wautersia eutropha (PhaC We ) has a subunit molecular weight of 64 kDa (8). The class III synthase from the prototypical Allochromatium Vinosum is composed of two subunits, designated PhaE Av (MW ≈ 40 kDa) and PhaC Av (MW ≈ 39 kDa) (9). PhaC Av contains the active si...

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“…For plant starch biosynthesis, the nature of a relevant primer remains elusive (44). Also for bacterial glycogen synthases, the requirement for a primer is controversial: E. coli GlgA appears to require a (1,4)-␣-glucan oligosaccharide as primer (45), whereas A. tumefaciens GlgA appears to be involved in both initiation and elongation of glycogen, without the need for a primer (33). In this study, we have shown for the first time that S. pombe cells contain a primer for (1,4)-␣-glucan biosynthesis (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…We tested glycogen synthase II from S. cerevisiae (Gsy2p) as a reporter, which normally elongates (1,4)-␣-glucan primers generated by glycogenin, but lacks the ability to initiate de novo (1,4)-␣-glucan synthesis (33,40). Overexpression of wildtype S. cerevisiae Gsy2p in S. pombe cells resulted in a brown staining, whereas low expression did not (Fig.…”

Section: The Intracellular Domain Of Ags1p Is Responsible For (14)-␣mentioning

confidence: 99%

Role of the Synthase Domain of Ags1p in Cell Wall α-Glucan Biosynthesis in Fission Yeast

Vos

Dekker

Distel

et al. 2007

Journal of Biological Chemistry

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The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides ␤-glucan and chitin, ␣-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall ␣-glucan is an essential component, consisting mainly of (1,3)-␣-glucan with ϳ10% (1,4)-linked ␣-glucose residues. The multidomain protein Ags1p is required for ␣-glucan biosynthesis and is conserved among cell wall ␣-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-␣-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-␣-glucan constituent of cell wall ␣-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-␣-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-␣-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-␣-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall ␣-glucan.

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De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation

Cited by 84 publications

References 20 publications

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

Class III Polyhydroxybutyrate Synthase: Involvement in Chain Termination and Reinitiation

Role of the Synthase Domain of Ags1p in Cell Wall α-Glucan Biosynthesis in Fission Yeast

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