Supplementary Figure 3: Tolerance of different OGT isoforms for single amino acid substitutions.
From: The active site of O-GlcNAc transferase imposes constraints on substrate sequence
OGT activity on the reference peptide KKVPVSRA was measured with two different constructs of the enzyme possessing a different number of TPR repeats. Nucleocytoplasmic OGT (ncOGT) is the longest (full length) natural OGT isoform, whereas the truncated construct, OGT (312—1031), was used for crystallographic studies and library screening due to its increased stability. The reference peptide KKVPVSRA represents the optimal OGT hexapeptide sequon except for position −3, where Val was used in order to avoid a potential second O-GlcNAc acceptor. Two N-terminal Lys residues were added, in order to aid peptide solubility. Assay details are given in the Online Methods section. The average of three measurements is shown, with error bars depicting the s.e.m. Activity for each enzyme isoform was normalized to the reference peptide. Full-length human OGT was expressed and purified as described for the truncated construct in Schimpl, M. et al., Nat.Chem.Biol. 8: 969, 2012.
