Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase.
A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.
Université de Nantes, CNRS UMR 6286, UFIP, 2 rue de la Houssinière, Nantes cedex 03 F-44322, France.
David
D
Teze
Université de Nantes, CNRS UMR 6286, UFIP, 2 rue de la Houssinière, Nantes cedex 03 F-44322, France.
Franck
F
Daligault
Ecole Nationale Supérieure de Chimie de Rennes, CNRS, UMR 6226, 11 allée de Beaulieu, CS 50837, Rennes cedex 35708, France Université européenne de Bretagne, Rennes, France.
Vincent
V
Ferrières
Université de Nantes, CNRS UMR 6286, UFIP, 2 rue de la Houssinière, Nantes cedex 03 F-44322, France.
Yves-Henri
YH
Sanejouand
Université de Nantes, CNRS UMR 6286, UFIP, 2 rue de la Houssinière, Nantes cedex 03 F-44322, France charles.tellier@univ-nantes.fr.
Charles
C
Tellier
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D004187:Disaccharides / Q000138:chemical synthesis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D004187:Disaccharides; D000519:alpha-Galactosidase
evolution; glycosidase; mutagenesis; oligosaccharide; transglycosylation