Research & Teaching Faculty

Default Header Image

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

TitleEngineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts
Publication TypeJournal Article
Year of Publication2005
AuthorsMullegger, J, Jahn, M, Chen, HM, WARREN, RAJ, Withers, SG
JournalPROTEIN ENGINEERING DESIGN & SELECTION
Volume18
Pagination33-40
Date PublishedJAN
ISSN1741-0126
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.

DOI10.1093/protein/gzi003