Distinct properties of Halobacterium salinarum Agl32, an archaeal D-glucuronyl C5-epimerase involved in N-glycosylation.

The N-linked tetrasaccharide decorating glycoproteins of the halophilic archaea Halobacterium salinarum offered the first example of N-glycosylation outside the Eukarya and still represents the only known instance of iduronic acid (IdoA) being employed in this post-translational modification. Recent identification of Agl32 as the D-glucuronyl C5-epimerase catalyzing the conversion of glucuronic acid (GlcA) into IdoA allows for comparing this enzyme in each of the three domains of life, namely, Eukarya, Bacteria and Archaea. Specifically, the current study assessed whether Agl32 requires flanking sugars on either side of the target GlcA, as do its eukaryal and bacterial counterparts. Nuclear magnetic resonance analysis of the glycan from an Hbt. salinarum mutant unable to add the fourth and final N-linked tetrasaccharide sugar revealed that Agl32 requires GlcA on both sides of the target GlcA for the epimerization reaction. Despite similar requirements for flanking sugars, Agl32 shares little structural similarity with eukaryal GlcE or DSepi1, D-glucuronyl C5-epimerases respectively involved in IdoA generation in the glycosaminoglycans heparin/heparan sulfate and dermatan sulfate. Moreover, Agl32 processes a substrate far shorter than what is recognized by the eukaryal enzymes. Indeed, it would appear that Agl32 relies on a catalytic mechanism distinct from that employed by these other D-glucuronyl C5-epimerases. Finally, the presence of agl32 homologues in putative N-glycosylation gene clusters in other haloarchaea argues that the use of IdoA in N-glycosylation extends beyond Hbt. salinarum.