Enhanced transfer of a photocrosslinking GlcNAc analog by an O-GlcNAc transferase mutant with converted substrate specificity [Enzymology]

August 3rd, 2015 by Rodriguez, A. C., Yu, S.-H., Li, B., Zegzouti, H., Kohler, J. J.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins in multi-cellular organisms. O-GlcNAc modification is catalyzed by the O-GlcNAc transferase (OGT), which transfers N-acetylglucosamine (GlcNAc) from the nucleotide sugar donor UDP-GlcNAc to serine or threonine residues of protein substrates. Recently, we reported a novel metabolic labeling method to introduce the diazirine photocrosslinking functional group onto O-GlcNAc residues in mammalian cells. In this method, cells are engineered to produce diazirine-modified UDP-GlcNAc (UDP-GlcNDAz), and the diazirine-modified GlcNAc analog (GlcNDAz) is transferred to substrate proteins by endogenous OGT, producing O-GlcNDAz. O-GlcNDAz-modified proteins can be covalently crosslinked to their binding partners, providing information about O-GlcNAc-dependent interactions. The utility of the method was demonstrated by crosslinking highly O-GlcNAc-modified nucleoporins to proteins involved in nuclear transport. For practical application of this method to a broader range of O-GlcNAc-modified proteins, efficient O-GlcNDAz production is critical. Here we examined the ability of OGT to transfer GlcNDAz and found that the wild-type enzyme (wtOGT) prefers the natural substrate, UDP-GlcNAc, over the unnatural UDP-GlcNDAz. This competition limits O-GlcNDAz production in cells and the extent of O-GlcNDAz-dependent crosslinking. Here we identified an OGT mutant, OGT(C917A) that efficiently transfers GlcNDAz and, surprisingly, has altered substrate specificity, preferring to transfer GlcNDAz rather than GlcNAc to protein substrates. We confirmed the reversed substrate preference by determining the Michaelis-Menten parameters describing the activity of wtOGT and OGT(C917A) with both UDP-GlcNAc and UDP-GlcNDAz. Use of OGT(C917A) enhances O-GlcNDAz production, yielding improved crosslinking of O-GlcNDAz-modified molecules both in vitro and in cells.