Structure and hydration of the amylopectin trisaccharide building blocks—Synthesis, NMR, and molecular dynamics

Pb, Hansen; Larsen, Flemming H.; Motawia, Mohammed Saddik; Blennow, Andreas; Spraul, Manfred; Dvortsák, Péter; Engelsen, Søren B.

doi:10.1002/bip.21075

Cited by 29 publications

(24 citation statements)

References 45 publications

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“…The third set of signals displayed chemical shifts of a bridging glucose unit that is ␣1,6-linked on either side. Chemical shifts of Glc-␣1,6Glc-␣1,6Glc reported previously (24,25) fitted perfectly the experimental data of the terminal and bridging glucoses, providing strong evidence for ␣1,6-linked glucose residues (supplemental Table 1). Chemical shifts calculated with the algorithm CASPER (26) further supported the assignment.…”

Section: A Pleuropneumoniae and Y Enterocolitica Hmw1csupporting

confidence: 76%

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Schwarz

Fan

Schubert

et al. 2011

Journal of Biological Chemistry

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N-Linked glycosylation is a frequent protein modification that occurs in all three domains of life. This process involves the transfer of a preassembled oligosaccharide from a lipid donor to asparagine side chains of polypeptides and is catalyzed by the membrane-bound oligosaccharyltransferase (OST). We characterized an alternative bacterial pathway wherein a cytoplasmic N-glycosyltransferase uses nucleotide-activated monosaccharides as donors to modify asparagine residues of peptides and proteins. N-Glycosyltransferase is an inverting glycosyltransferase and recognizes the NX(S/T) consensus sequence. It therefore exhibits similar acceptor site specificity as eukaryotic OST, despite the unrelated predicted structural architecture and the apparently different catalytic mechanism. The identification of an enzyme that integrates some of the features of OST in a cytoplasmic pathway defines a novel class of N-linked protein glycosylation found in pathogenic bacteria.N-Linked glycosylation is characterized by an N-glycosidic linkage between the side chain amide of an asparagine residue of proteins and an oligosaccharide. This type of glycosylation occurs in both prokaryotes and eukaryotes and requires the assembly of an oligosaccharide on a polyisoprenoid lipid by sequential addition of monosaccharides, catalyzed by cytosolic glycosyltransferase. The resulting lipid-linked oligosaccharide is translocated to the luminal side of the endoplasmic reticulum membrane or the plasma membrane of prokaryotes, where it may be further elongated. The glycan is then transferred to the ␦-amino group of asparagine residues within the consensus sequence NX(S/T) of polypeptides. This reaction is catalyzed by oligosaccharyltransferase (OST), 4 a membrane-bound enzyme that can be composed of several different subunits (1). As oligosaccharide transfer takes place in the endoplasmic reticulum or in the periplasm, N-glycosylation affects proteins trafficking along the secretory pathway.N-Glycosylation exhibits important physiological functions. In the early secretory pathway of eukaryotes, N-glycans present on newly synthesized proteins orchestrate the folding of glycoproteins and act as a signal for directing misfolded polypeptides to degradation (2). After being processed in the Golgi organelle, N-linked glycans are relevant, among other processes, for the modulation of the immune system and for the control of immune cell homeostasis and inflammation (3, 4). The importance of this protein modification is supported by its incidence: more than half of all eukaryotic proteins are glycosylated (5).About 8 years ago, a study uncovered that the extracellular HMW1A adhesin of the Gram-negative bacterium Haemophilus influenzae is modified with hexose monosaccharides on asparagine residues (6). The pioneering work of St Geme and co-workers (7, 8) subsequently showed that the modified asparagine residues are found within the same consensus sequence recognized by OST (i.e. NX(S/T)) and that the enzyme responsible for this modification is the HMW1C p...

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Section: A Pleuropneumoniae and Y Enterocolitica Hmw1csupporting

confidence: 76%

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Schwarz

Fan

Schubert

et al. 2011

Journal of Biological Chemistry

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“…C6 phosphorylation triggers C3 phosphorylation by phosphoglucan water dikinase (PWD) (8,10,11). Recent data suggest that C6 phosphorylation fits within the unphosphorylated structure of the amylopectin helix, but C3 phosphorylation imposes significant steric effects and is predicted to induce a conformational change (12)(13)(14)(15). This suggests that GWD-directed C6 phosphorylation promotes local hydration of crystalline lamellae and that PWD-directed C3 phosphorylation induces helix strain.…”

mentioning

confidence: 99%

Structural basis for the glucan phosphatase activity of Starch Excess4

Kooi

Taylor

Pace

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydratebinding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.carbohydrate | Lafora disease | laforin | phosphorylation P lants and animals store carbohydrates as starch and glycogen, respectively. Starch is produced in diurnal cycles and is composed of <10% w∕w amylose and >80% w∕w amylopectin in Arabidopsis thaliana leaves (1). Amylose is a linear molecule composed of glucose moieties linked by α-1,4-glycosidic linkages with very few branches. Amylopectin, which is similar to glycogen, is composed of α-1,4-glycosidic linkages with α-1,6-glycosidic branches, but amylopectin branches are arranged in clusters at regular intervals and the branches form double helices that pack together to form crystalline lamellae (2, 3). The decreased branching and crystalline lamellae of amylopectin are key contributors to the insolubility of starch, while glycogen has more branches and is water-soluble.Starch is a water-insoluble polymer whose surface is inaccessible to most enzymes. Recent work convincingly demonstrates that reversible starch phosphorylation and dephosphorylation is essential for processive starch metabolism (reviewed in refs. 4-7). An essential signal triggering starch catabolism is phosphorylation on the C6 position of glucose moieties on the surface of starch by glucan water dikinase (GWD/R1) (8, 9). C6 phosphorylation triggers C3 phosphorylation by phosphoglucan water dikinase (PWD) (8,10,11). Recent data suggest that C6 phosphorylation fits within the unphosphorylated structure of the amylopectin helix, but C3 phosphorylation imposes significant steric effects and is predicted t...

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“…However, in panose, the first and second glucose residues (from the nonreducing end) are linked with an ␣-1,6 glycosidic bond. In isopanose, the ␣-1,6 linkage is located between the two glucose residues at the reducing end (25). The E. faecalis strain V583 has been shown to grow on panose (26), and several other bacteria were reported to utilize both panose and isopanose (27)(28)(29).…”

Section: Substratementioning

confidence: 99%

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake

et al. 2017

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Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of ␣-1,4-and some ␣-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6Љ-P in the cell. MapP, which dephosphorylates maltose-6=-P, also releases P i from maltotriose-6Љ-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection. IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics, and new strategies are urgently needed. A largely unexplored aspect is how these pathogens proliferate and which substrates they use in order to grow inside infected hosts. The use of maltodextrins as a source of carbon and energy was studied in Enterococcus faecalis and linked to its virulence. Our results demonstrate that E. faecalis can efficiently use glycogen degradation products. We show here that depending on the length of the maltodextrins, one of two different

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Structure and hydration of the amylopectin trisaccharide building blocks—Synthesis, NMR, and molecular dynamics

Cited by 29 publications

References 45 publications

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Cytoplasmic N-Glycosyltransferase of Actinobacillus pleuropneumoniae Is an Inverting Enzyme and Recognizes the NX(S/T) Consensus Sequence

Structural basis for the glucan phosphatase activity of Starch Excess4

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake

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