Identification of residues important for the activity of Haloferax volcanii AglD, a component of the archaeal N-glycosylation pathway.
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.
Kaminski
Lina
L
Department of Life Sciences, Ben Gurion University, Beersheva, Israel.
Eichler
Jerry
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D019622:Haloferax volcanii / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D019843:Archaeal Proteins; D008562:Membrane Glycoproteins; D050505:Mutant Proteins; C055764:S-layer proteins; D005964:Glucosyltransferases