Abstract Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine transferase (EOGT), a glycosyltransferase (GT)-61 family member, catalyzes O-N-acetylglucosamine (GlcNAc) transfer from UDP-GlcNAc to serine or threonine residues within EGF domains in the endoplasmic reticulum. In this study, we determined the crystal structure of the EOGT–uridine diphosphate (UDP) complex and identified the critical residues mediating their interactions, which were validated via site-directed mutagenesis and enzyme activity assays. These residues were conserved in EOGT orthologs across metazoans, and UDP binding occurred independently of divalent metal ions and the canonical Asp–X–Asp motif. Although EOGT catalyzes O-GlcNAcylation, similar to O-GlcNAc transferase (OGT), it shares little sequence similarity with OGT and belongs to a distinct glycosyltransferase family. Instead, EOGT is more closely related to protein O-linked-mannose β1,4-N-acetylglucosaminyltransferase 2 (POMGNT2). Structural comparison with POMGNT2 revealed a conserved triad of one asparagine and two arginine residues, the N–R–R constellation. These elements were conserved across metazoans and green plants (Viridiplantae), suggesting a unifying mechanism of UDP recognition and providing a framework to interpret disease-associated EOGT mutations and assess the evolution of catalytically active GT61 family enzymes.
Tashima et al. (Wed,) studied this question.