Oligosaccharide Binding in Escherichia coli Glycogen Synthase

Abstract

Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

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

“…The data presented here are consistent with what has been found for the bacterial and archaeal glycogen synthases (12,13) and glycogen phosphorylase (26) in that they integrate their carbohydrate-binding sites onto the surface of the enzyme. The physical and chemical features of the sites are comparable with the B-type carbohydrate-binding modules, which are characterized by the presence of a hydrophobic cleft or groove that accommodates at least two sugar moieties where additional points of contact are mediated through hydrogen bonding to the polar edges of the sugar molecules (25).…”

“…Based on these results, it was proposed that the enzyme has two distinct acceptor substrate-binding sites, a catalytic site and a polysaccharide-binding site, and occupancy of both sites is required for high catalytic efficiency (11). Consistent with this hypothesis, structural * This work was supported, in whole or in part, by National Institutes of Health Grants R37-DK027221 and R01-NS056454 (to P. J. R.), R01-DK079887 (to T. D. H.), and R15-GM081810 (to W. A. W. studies of the Escherichia coli (12) and Pyrococcus abyssi (13) glycogen synthases have identified oligosaccharide-binding sites in the N-terminal domain of the respective enzymes that impact catalysis on glycogen in both the archaeal and human enzymes (13). To examine the mechanism by which eukaryotic glycogen synthases bind their substrates, we solved the structures of yeast Gsy2p in association with maltooctaose in both its basal (R580A/R581A/R583A) and activated (R589A/R592A with glucose 6-phosphate) state conformations.…”

“…These structures reveal the presence of four distinct maltodextrin-binding sites on the surface of the enzyme. One of the maltodextrin-binding sites is similar in general location to sites observed in the P. abyssi and E. coli enzymes (12,13) but is formed from structural elements unique to the eukaryotic enzymes (residues 117-121, 145-156, and 182-184). The remaining three sites (site-2 (residues 365-367 and 439 -465), site-3 (residues 333-340 and 461-463) and site-4 (residues 208 -211, 324, and 507-561)) are found on the surface of the C-terminal domain and have no known counterpart in the bacterial or archaeal enzymes.…”

“…In both the P. abyssi and E. coli enzymes, all of the glycogenbinding sites are distributed on the surface of the N-terminal domain, where it was proposed, for the E. coli enzyme, that they efficiently uncouple substrate association from the domain movement necessary for catalysis (12). Unlike these enzymes, yeast Gsy2p has binding sites located on both the N-and C-terminal domains, and sequence insertions that form secondary structural elements unique to the eukaryotic enzymes contribute to these binding sites.…”

Identification and characterization of granule bound starch synthase I (GBSSI) gene of tartary buckwheat (Fagopyrum tataricum Gaertn.)

“…6). As in Escherichia coli glycogen synthase (EcGS; Sheng et al, 2009b) and Oryza sativa japonica GBSSI (OsGBSSI; Momma and Fujimoto, 2012) structures, the N-and C-terminal domains of FtGBSSI "close" and form a catalytic cleft. The two domains are connected through a narrow hinge peptide, as well as the C-terminal domain and C-terminal helix.…”