PMID:15790578
| Citation |
Müllegger, J, Jahn, M, Chen, HM, Warren, RA and Withers, SG (2005) Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts. Protein Eng. Des. Sel. 18:33-40 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1093/protein/gzi003 |
| Keywords |
Acids/metabolism; Base Sequence; Catalysis; DNA Primers; Glycosylation; Glycosyltransferases/chemistry; Glycosyltransferases/genetics; Glycosyltransferases/metabolism; Hydrogen-Ion Concentration; Kinetics; Mutagenesis; Nuclear Magnetic Resonance, Biomolecular; Protein Engineering |
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