Characterisation of the activator effect of glycogen on amylosucrase from Neisseria polysaccharea

International audienc

Glucansucrases: molecular engineering and oligosaccharide synthesis

International audienc

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

International audienc

Site-directed mutagenesis of key amino acids in the active site of amylosucrase from Neisseria polysaccharea

International audienceAmylosucrase from Neisseria polysaccharea (AS) is a glucosyltransferase from family 13 of the glycoside hydrolases. In this family, AS shows an unusual specificity for sucrose, which is the best substrate for the enzyme. AS synthesises, from this high-energy substrate, an amylose-like polymer. In addition, it catalyses the transfer of glucose units from sucrose onto acceptor molecules like glucose, maltooligosaccharides or glycogen. Finally, it catalyses the disproportionation of maltooligosaccharides. A structural analysis of a mutated AS complexed with sucrose led to a detailed description of the active site and of the interactions between sucrose and AS at subsites -1 and + 1. Site-directed mutagenesis experiments confirmed the essential role of residues always conserved in the alpha-amylase family. The nucleophile Asp286 and the general acid-base catalyst Glu328 are identified unequivocally. The conserved residues Asp393, His187, His 392, Arg284 and the stacking residues Tyr147 and Phe250 are critical for the enzymatic activity. These results support, for AS, an alpha-retaining mechanism via a double-displacement, similar to that described for alpha-amylases. In addition, the salt bridge formed by Asp144 and Arg509 is essential for the architecture of the active site and consequently for the sucrose specificity. Finally, Asp394 and Arg446 which interact with the fructosyl ring are not essential for activity towards sucrose but could be crucial for the binding of acceptor molecules

crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Glm in complex with the natural substrate sucrose

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Crystallization and preliminary X-ray studies of recombinant amylosucrase from Neisseria polysaccharea

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Amylosucrase, a glucan-synthesizing enzyme from the α-amylase family

Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the α-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (β/α)8-barrel A-domain, B- and B′-domains displaying α/β-structure, and a C-terminal eight-stranded β-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (β/α)8-barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B′) is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B′-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket