Agl24 is an ancient archaeal homolog of the eukaryotic N-glycan chitobiose synthesis enzymes.

Protein N-glycosylation is a post-translational modification found in organisms of all domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in Eukaryotes, although the enzyme catalyzing this reaction remains unknown. Here, we report the identification of a thermostable archaeal β-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed its function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed similarities of Agl24 to the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which are catalyzing the same or a similar reaction, respectively. Phylogenetic analysis of Alg14/13 homologs indicates that they are ancient in Eukaryotes, either as a lateral transfer or inherited through eukaryogenesis.