Rational Design of an Aryl-C-Glycoside Catalyst from a Natural Product O-Glycosyltransferase

SummaryBecause the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (197 K)Download as PowerPoint slideHighlights► 106 rationally designed chimeras were expressed in vivo and screened by HPLC-MS analysis ► One 12-amino acid protein segment is crucial for O- or C-glycosylation ► The sugar-acceptor postioning determines O- or C-glycosidic bond formation ► Ala substitutions revealed the key spots for glycosylation