title: Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase.
abstract: A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.
authors.affiliation: Université de Nantes, CNRS FRE n°3478, UFIP, 2 rue de la Houssinière, F-44322 Nantes cedex 03, France.
authors.forename: David
authors.initials: D
authors.lastname: Teze
authors.forename: Johann
authors.initials: J
authors.lastname: Hendrickx
authors.forename: Mirjam
authors.initials: M
authors.lastname: Czjzek
authors.forename: David
authors.initials: D
authors.lastname: Ropartz
authors.forename: Yves-Henri
authors.initials: YH
authors.lastname: Sanejouand
authors.forename: Vinh
authors.initials: V
authors.lastname: Tran
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
authors.forename: Michel
authors.initials: M
authors.lastname: Dion
mesh_terms: D019295:Computational Biology; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics / Q000502:physiology; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase
keywords: glycosidase; mutagenesis; oligosaccharide; structure; transglycosylation