Control of glycosyltransferase activity by nucleotide-binding active site residues in SECRET AGENT.

Human O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the only glycosyltransferase that operates in the nucleus and cytoplasm, targeting over 1,000 unique substrates. OGT is conserved across all eukaryotic species, and misregulation of protein O-GlcNAcylation has been implicated in cancers and neurodegenerative diseases. Secret Agent (SEC) is a structural homolog of OGT in Arabidopsis thaliana and like OGT, it consists of two comparably-sized domains: an N-terminal tetratricopeptide repeat (TPR) domain that mediates protein-protein interactions and a C-terminal catalytic domain that mediates O-GlcNAc transfers. Both SEC and OGT transfer GlcNAc from UDP-GlcNAc to serine and threonine residues on their substrates, but the exact mechanisms of SEC nucleotide-sugar selection and catalysis are unknown. In the catalytic domain, UDP-GlcNAc binds directly to OGT and makes several key contacts to active site residues before protein substrates bind over the nucleotide-sugar, making nucleotide-sugar binding essential for catalysis. To determine whether OGT's mechanisms of nucleotide-sugar selection and catalysis are conserved in the SEC homolog, we investigated residues in the SEC active site corresponding to known essential residues in OGT that make key interactions with the UDP nucleotide portion of UDP-GlcNAc. Mutant SEC protein was purified from E. coli and analyzed for catalytic activity. Active site mutations known to inhibit OGT activity were also found to reduce activity in SEC, suggesting a conserved mechanism for nucleotide-sugar selection.