The Crystal Structures of the Open and Catalytically Competent Closed Conformation of Escherichia coli Glycogen Synthase

Abstract

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2°o verall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the ␣-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site.

Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

“…A similar result was observed in the complexes between ADP-Glc and EcGS (24), and between UDP-Glc and Arabidopsis thaliana sucrose synthase, a member of the retaining GT4 family (19). In those cases, however, the respective diphosphonucleoside moieties of the donor molecule were present in the active sites of the enzymes, although not covalently bound to the not precisely identified glucose derivative.…”

“…A strong H-bond to the Arg152 backbone amide would result in an increased basicity of the carbonyl oxygen of His151, which in turn would favor the capture of the C2 proton. Asp128 is absolutely conserved in all members of family GT5 (24) and in the equivalent position eukaryotic GSs have a glutamic acid residue. In yeast GS2, the side chain carboxylate of this residue (Glu169), which also has a disallowed Ramachandran conformation, is also within hydrogen bonding distance of the backbone amide protons of the essential His193 (equivalent to His151 in PaGS) and the next residue, Ala194.…”

“…Fungal and animal GSs, classed in family GT3, have only one representative, Saccharomyces cerevisiae GS, of known crystal structure (21), while three enzymes of the GT5 family, which comprises prokariotyc and archaeal GSs, as well as starch synthases, have been solved: those from Agrobacterium tumefaciens (22), Pyrococcus abyssi glycogen synthase (PaGS) (23), and Escherichia coli glycogen synthase (EcGS) (24). The crystal structure of the complex between EcGS and ADP-Glc showed that the enzyme is able to cleave its donor substrate into ADP and an unknown glucosyl species.…”

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

“…In a previous research, four sugar (glycan) binding sites were identified in EcGS (Sheng et al, 2009a). The first one was found buried in the interdomain active site cleft, corresponding to three well-defined sugar (glucose) moieties extending from the active site toward the enzyme surface, whereas the other three sites were observed at the Nterminal domain surface.…”

“…The first one was found buried in the interdomain active site cleft, corresponding to three well-defined sugar (glucose) moieties extending from the active site toward the enzyme surface, whereas the other three sites were observed at the Nterminal domain surface. The sugar units at the first site are strongly associated to the active site, and amino acid residues are highly conserved throughout the GS, SS, MalP (maltodextrin phosphorylase), and GP families (Sheng et al, 2009a). Thanks to this information, we therefore predicted the corresponding residues involved in the first sugar binding site in FtGBSSI.…”