Figure 5. The OTase AglB utilizes Dol-P-disaccharide to generate an N-linked glycopeptide.

(a) AglB catalyzes the transfer of the disaccharide GlcNAc-Glc-2,3-diNAcA from the AglC product Dol-P-GlcNAc-Glc-2,3-diNAcA to an acceptor peptide. (b) AglB activity assay using the peptide YKYNESSYK, based on the M. voltae flagellum protein FlaB2, as the acceptor substrate and Dol-P-GlcNAc (MS) or Dol-P-disaccharide (DS) as the donor substrates. A peptide lacking the key asparagine residue, YKYQESSYK, was also screened. (c) AglB assay performed with either the addition of MnCl₂, no exogenous MnCl₂, or EDTA. Assay data shown in (b,c) are representative traces from at least three independent experiments. Variability seen between runs was less than 5%. (d) RP-HPLC purification of the peptide and the glycopeptide (280 nm).