The active site of the Escherichia coli glycogen synthase is similar to the active site of retaining GT-B glycosyltransferases q Alejandra Yep, Miguel A. Ballicora, and Jack Preiss * Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA Received 20 February 2004 Abstract Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-re- ducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific ac- tivity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the K m for glycogen and only H161A and R300A had a higher K m for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Glycogen; Glycosyltransferase; Glycogen synthase; Maltodextrin phosphorylase; Retaining configuration; glgA The biosynthesis of complex carbohydrates and polysaccharides is of remarkable biological importance. These molecules are involved in a large series of cellular processes, including energy storage, building of cell wall structure, signaling, cell–cell interaction. and post- translational modification of proteins. The superfamily of glycosyltransferases catalyzes the transfer of a prop- erly activated sugar residue to an acceptor molecule, which can be a lipid, a protein or another carbohydrate. The glycosylation reaction is highly specific with respect to the anomeric configuration of the sugar residue and the site of addition [1]. Glycosyltransferases have been classified in three different ways, according to: (i) sequence similarity, (ii) the anomeric configuration of the product, and (iii) the protein fold. The glycosyltransferase superfamily is di- vided into 69 families (GT1–GT69), based on high-se- quence similarity to one or more founding members with experimentally demonstrated enzymatic activity (http://afmb.cnrs-mrs.fr/CAZY/index.html) [2]. Two types of reactions have been described, retaining, and inverting, depending on whether the anomeric configu- ration of the sugar donor is the same or different from that of the product. For these two reactions, two main catalytic mechanisms have been proposed. In the in- verting mechanism, the acceptor is thought to perform a nucleophilic attack at C1 of the sugar donor changing its anomeric configuration [1]. The details of the retaining mechanism are less clear, but it has been proposed that a nucleophilic substitution proceeds through a double displacement mechanism, involving a late oxonium-ion- like transition state [3,4]. In addition, these enzymes can be grouped according to their protein fold, namely, GT- A, GT-B, and GT-C [5]. The glycogen synthase (EC 2.4.1.21) from Escherichia coli belonging to family GT5 is a retaining glycosyl- transferase, and it has a putative GT-B fold based on secondary structure predictions and threading [6]. q Abbreviations: ADP-Glc, ADP-glucose; Glc1P, glucose 1-phos- phate; Glc6P, glucose 6-phosphate; MalP, maltodextrin phosphorylase; MurG, undecaprenyldiphospho-muramoylpentapeptide b-N-acetylglu- cosaminyltransferase; OtsA, trehalose-6-phosphate synthase; UDP-Glc, UDP-glucose. * Corresponding author. Fax: 1-517-353-9334. E-mail address: preiss@msu.edu (J. Preiss). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.136 Biochemical and Biophysical Research Communications 316 (2004) 960–966 BBRC www.elsevier.com/locate/ybbrc