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Volume 2010, Article ID 315108, 9 pages
Research Article

Identification of Residues Important for the Activity of Haloferax volcanii AglD, a Component of the Archaeal N-Glycosylation Pathway

Department of Life Sciences, Ben Gurion University, P. O. Box 653, Beersheva 84105, Israel

Received 11 January 2010; Accepted 10 February 2010

Academic Editor: Julie Maupin-Furlow

Copyright © 2010 Lina Kaminski and Jerry Eichler. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.