Biologia, Bratislava, 57/Suppl. 11: 119-128, 2002.
ISSN 0006-3088 (Biologia).
Site-directed mutagenesis of key amino acids in the active site of amylosucrase from Neisseria polysaccharea.
Cecile Albenne1, Gabrielle Potocki de Montalk1, Pierre Monsan1, Lars Skov2, Osman Mirza2, Michael Gajhede2 & Magali Remaud-Simeon1*
1 Centre de Bioingenierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA 135, avenue de Rangueil, F-31077 Toulouse, France; tel.: ++ 335 61 55 94 15, fax: ++ 335 61 55 94 00, e-mail: email@example.com
2 Protein Structure Group, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
Received: October 17, 2001 / Accepted: February 12, 2002
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 a-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 a-retaining mechanism via a double-displacement, similar to that described for a-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.
Key words: amylosucrase, sucrose, active site, a-amylase, site-directed mutagenesis, substrate specificity.