In Haloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in an Hfx. volcanii aglD deletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme. 1. Introduction Although the presence of N-glycosylated proteins in Archaea has been known for over 30 years [1], the pathways responsible for this posttranslational modification have only recently been addressed. In Methanococcus voltae, Methanococcus maripaludis, and Haloferax volcanii, products of the agl genes have been shown to participate in the assembly of oligosaccharides decorating various glycoproteins in these species [2–4]. At present, however, apart from the oligosaccharyltransferase, AglB [5–7], virtually nothing is known of the catalytic workings of the different Agl proteins. Of the Hfx. volcanii Agl proteins identified to date, at least five (i.e., AglD, AglE, AglG, AglI, and AglJ) are predicted to act as glycosyltransferases (GTs), enzymes that catalyze the formation of glycosidic bonds through the transfer of the sugar moieties from nucleotide-activated saccharides to appropriate targets [8]. Based on their amino acid similarities, GTs can be classified into 91 family groups (http://www.cazy.org/fam/acc_GT.html; January, 2009), varying in size and number of functions fulfilled by family members [9, 10]. Furthermore, the different GT families can be clustered based on whether the canonical GT-A or GT-B fold is employed and whether sugar stereochemistry is retained or inverted upon addition of a glycosyl donor [11]. Still, the ability to predict the function of a given GT or to define its catalytic mechanism remains a challenge. This is particularly true in the case of the GT2 family, an ancient group of GT-A
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