Skip Navigation



PEDS Advance Access published online on November 19, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp068
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
23/1/43    most recent
gzp068v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Tran, V.
Right arrow Articles by Dion, M.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tran, V.
Right arrow Articles by Dion, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Rational design of a GH1 β-glycosidase to prevent self-condensation during the transglycosylation reaction

Vinh Tran, Lionel Hoffmann, Claude Rabiller, Charles Tellier and Michel Dion1

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

1 To whom correspondence should be addressed. E-mail: michel.dion{at}univ-nantes.fr

Mutant N282T of a thermostable β-glycosidase from GH1 family (TtβGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-β-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.

Keywords: β-glycosidase/enzyme–substrate modeling/N-acetyl-glucosamine/oligosaccharide/transglycosylation

Received July 23, 2009; revised October 16, 2009; accepted October 20, 2009.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.