title: Rational design of a GH1 beta-glycosidase to prevent self-condensation during the transglycosylation reaction.
abstract: Mutant N282T of a thermostable beta-glycosidase from GH1 family (TtbetaGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-beta-d-galactopyranoside (oNPGal) which competes with the condensation reaction and entails undesirable effects. In order to prevent this reaction, we rationally modified this enzyme at the [+1]/[+2] subsites so that oNPGal would bind less tightly. Molecular modeling (MM) suggested the mutation A221W, which decreased the affinity of the donor at these sites and moved it away from the bound galactose at the -1 subsite. A single (A221W) and a double mutant (A221W/N282T) were constructed, and they gave rise to a drastic decrease in self-condensation. The A221W mutant had no transglycosylation activity whereas the A221W/N282T mutant still displayed a condensation activity, comparable to that of the N282T mutant for the transfer on pNPGlcNAc. MM revealed that the double mutant A221W/N282T could induce the synthesis of a glycosidic bond between a donor and an acceptor displaying an equatorial 4-position. Moreover, it is suggested that mutation N282T could change the orientation of residue N219, leading to a stabilization of the acceptor with a new hydrogen bond. This finding opens the way to further improvements of evolved transglycosidases.
authors.affiliation: Faculté des Sciences et des Techniques, Biotechnologie, Biocatalyse, Biorégulation (UMR CNRS 6204) Nantes University, PRES UNAM, 2 rue de la Houssinière, BP 92208, F-44322 Nantes cedex 3, France.
authors.forename: Vinh
authors.initials: V
authors.lastname: Tran
authors.forename: Lionel
authors.initials: L
authors.lastname: Hoffmann
authors.forename: Claude
authors.initials: C
authors.lastname: Rabiller
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
authors.forename: Michel
authors.initials: M
authors.lastname: Dion
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D019020:Directed Molecular Evolution; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015202:Protein Engineering; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics; D014166:Transferases / Q000737:chemistry*
chemical_list: D009097:Multienzyme Complexes; C023176:transglycosidase enzyme system; D014166:Transferases; D006026:Glycoside Hydrolases