Identification and Characterization of EmNag, a Novel β-1,2-N-Acetylglucosaminidase from Elizabethkingia meningoseptica.

N-Acetylglucosamine exists in various glycoforms and linkage patterns within organisms, playing a crucial role in numerous vital biological processes. The signal of β-1,2-N-acetylglucosamine is a key component within complex biantennary N-glycans on the cell surface, leaving related research in its early exploratory stages. Currently, there is a lack of efficient tools and methods capable of directly modifying terminal N-acetylglucosamine on living cells. In this study, we identified a novel β-N-acetylglucosaminidase from Elizabethkingia meningoseptica FMS-007, which specifically cleaves nonreducing terminal β-1,2-N-acetylglucosamine, enabling direct removal from native glycoproteins and/or free oligosaccharides. More importantly, we demonstrate for the first time the successful removal of β-1,2-N-acetylglucosamine from the living cell surface using this enzyme. Based on its isolation and newly characterized cell-surface glycan-editing activity, we designated it as EmNag, also termed cell-surface glycan-editing N-acetylglucosaminidase (csgeNag). It showed favorable enzymatic stability between pH 5-8 and from 4 to 37 °C. Sequence analysis and alanine-scanning mutagenesis identified a conserved active pocket containing the catalytic residue pair Asp317-Glu318, with Asp317 being essential for activity. In summary, our findings provide an effective and reliable method for the targeted removal of β-1,2-N-acetylglucosamine from the living cell surface, establishing a foundation for further functional studies and practical applications.