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

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Cécile Albenne, Gabrielle Potocki De Montalk, Pierre Monsan, Lars Skov, Osman Mirza, Michael Gajhede, Magali Remaud-Simeon

Amylosucrase 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 α-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 α-retaining mechanism via a double-displacement, similar to that described for α-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.

Original languageEnglish
JournalBiologia - Section Cellular and Molecular Biology
Volume57
Issue numberSUPPL. 11
Pages (from-to)119-128
Number of pages10
ISSN1335-6399
Publication statusPublished - 1 Dec 2002

    Research areas

  • α-amylase, Active site, Amylosucrase, Site-directed mutagenesis, Substrate specificity, Sucrose

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