Mechanistic study of nucleotide-sugar binding and catalysis in SECRET AGENT O-GlcNAc Transferase.

O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential protein that is highly conserved across all eukaryotes. Misregulation of OGT's activity has been implicated in cancers and neurodegenerative diseases. OGT is unique in that it transfers a GlcNAc sugar from UDP-GlcNAc to over 1,000 nuclear and cytoplasmic proteins in almost every cellular process. The mechanisms of nucleotide-sugar binding and sugar transfer have been well characterized in human OGT. Secret Agent (SEC), an OGT structural homolog in Arabidopsis thaliana, also uses UDP-GlcNAc as its sugar donor, but the mechanisms of nucleotide-sugar binding and catalysis are not well characterized. We investigated how the SEC homolog selects UDP-GlcNAc, looking particularly at the residues that are hypothesized to interact with the GlcNAc portion of UDP-GlcNAc in OGT and drive deprotonation of protein substrates. However, it is unknown if the function of these residues is conserved in SEC. Active site residues in SEC corresponding to known essential residues in OGT were mutated using site-directed mutagenesis and expressed and purified from E. coli. Their catalytic activities were analyzed using glycosyltransferase activity assays and compared to the wildtype enzyme. These mutations led to lower relative activity, similar to OGT studies, suggesting that the binding and orientation mechanism of UDP-GlcNAc required for sugar transfer is conserved between SEC and OGT.