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PEDS Advance Access published online on March 14, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi003
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received November 18, 2004
Revised January 19, 2005
Accepted January 20, 2005

Article

Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts

Johannes Müllegger 1, Michael Jahn 1, Hong-Ming Chen 1, R. Antony J. Warren 2, and Stephen G. Withers 1*

1 Protein Engineering Network of Centres of Excellence, Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada
2 Protein Engineering Network of Centres of Excellence, Department of Microbiology, University of British Columbia, Vancouver, BC V6T 1Z1, Canada

* To whom correspondence should be addressed.
Stephen G. Withers, E-mail: withers{at}chem.ubc.ca


  Abstract

Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid-base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid-base mutant of Agrobacterium sp. {beta}-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid-base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.

Keywords: enzymatic oligosaccharide synthesis; {beta}-glucosidase; thioglycoligase; thioglycoside.
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