Fig. 1: Design of a xylosyltransferase BH system.
From: Xylosyltransferase engineering to manipulate proteoglycans in mammalian cells
a, Principle of the BH approach. WT-XTs transfer Xyl to substrate proteins that can be extended to GAG chains. BH-XTs transfer a bioorthogonal Xyl analog for visualization and MS profiling. b, Structural considerations of XT1 engineering to accept UDP-6AzGlc instead of UDP-Xyl. Insert, gatekeeper residues in the XT1 crystal structure (PDB 6EJ7) and structural trajectory of the azidomethyl modification in UDP-6AzGlc. c, In vitro glycosylation of a fluorescently labeled bikunin substrate peptide by WT-XT1 or mutant XT1 and different UDP-sugars (UDP-Xyl in black, UDP-Glc in light gray and UDP-6AzGlc in yellow). Data are individual data points, representing the mean ± s.d. from n = 3 technical replicates from one of two independent experiments. d, Michaelis–Menten kinetics of in vitro peptide glycosylation by different XT1–UDP-sugar combinations. Data are one independent replicate with n = 3 technical replicates, representing the average ± range from two independent replicates each or mean ± s.e.m. of n = 3 independent replicates (BH-XT1–UDP-Glc).
