title: Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts.
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.
authors.affiliation: Protein Engineering Network of Centres of Excellence, Department of Chemistry and Department of Microbiology, University of British Columbia, Vancouver, BC V6T 1Z1, Canada.
authors.forename: Johannes
authors.initials: J
authors.lastname: Müllegger
authors.forename: Michael
authors.initials: M
authors.lastname: Jahn
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Chen
authors.forename: R Antony J
authors.initials: RA
authors.lastname: Warren
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000143:Acids / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D015202:Protein Engineering
chemical_list: D000143:Acids; D017931:DNA Primers; D016695:Glycosyltransferases