title: Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis.
abstract: We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k(cat)/K(m) of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.
authors.affiliation: Pharmaceutical Sciences Division, School of Pharmacy, Wisconsin Center for Natural Products Research, University of Wisconsin, Madison, WI 53705-2222, USA.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Gantt
authors.forename: Pauline
authors.initials: P
authors.lastname: Peltier-Pain
authors.forename: Shanteri
authors.initials: S
authors.lastname: Singh
authors.forename: Maoquan
authors.initials: M
authors.lastname: Zhou
authors.forename: Jon S
authors.initials: JS
authors.lastname: Thorson
mesh_terms: D002384:Catalysis; D014644:Genetic Variation; D054794:Glycomics / Q000379:methods; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D057166:High-Throughput Screening Assays; D008968:Molecular Conformation; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry; D009702:Nucleoside Diphosphate Sugars / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic; D014539:Uridine Diphosphate Sugars / Q000737:chemistry*
chemical_list: D009596:Nitrophenols; D009702:Nucleoside Diphosphate Sugars; D014539:Uridine Diphosphate Sugars; C024719:4-nitrophenyl; D016695:Glycosyltransferases
keywords: carbohydrate; enzyme; glycobiology; protein engineering