Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation

• Published in: Nature communications Vol. 4; no. 1; pp. 2627 - 11
• Main Authors: Lizak, Christian, Gerber, Sabina, Michaud, Gaëlle, Schubert, Mario, Fan, Yao-Yun, Bucher, Monika, Darbre, Tamis, Aebi, Markus, Reymond, Jean-Louis, Locher, Kaspar P.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.10.2013; Nature Publishing Group
• Subjects: 101/6; 631/45/173; 631/45/603; 631/92/458/1524; 82; 82/16; 82/29; 82/58; Activation; Amides - metabolism; Asparagine; Asparagine - metabolism; Aspartic Acid - analogs & derivatives; Aspartic Acid - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Campylobacter jejuni - enzymology; Campylobacter jejuni - genetics; Campylobacter lari - enzymology; Campylobacter lari - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Expression; Glutamine; Glutamine - metabolism; Glycan; Glycopeptides; Glycosylation; Hexosyltransferases - chemistry; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; Homoserine - metabolism; Humanities and Social Sciences; Kinetics; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Models, Molecular; multidisciplinary; Oligosaccharyltransferase; Organic chemistry; Peptides; Protein Binding; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Science; Substrate Specificity; Twisting
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms3627

The initial glycan transfer in asparagine-linked protein glycosylation is catalysed by the integral membrane enzyme oligosaccharyltransferase (OST). Here we study the mechanism of the bacterial PglB protein, a single-subunit OST, using chemically synthesized acceptor peptide analogues. We find that PglB can glycosylate not only asparagine but also glutamine, homoserine and the hydroxamate Asp(NHOH), although at much lower rates. In contrast, N -methylated asparagine or 2,4-diaminobutanoic acid (Dab) are not glycosylated. We find that of the various peptide analogues, only asparagine- or Dab-containing peptides bind tightly to PglB. Glycopeptide products are unable to bind, providing the driving force of product release. We find no suitably positioned residues near the active site of PglB that can activate the acceptor asparagine by deprotonation, making a general base mechanism unlikely and leaving carboxamide twisting as the most likely mechanistic proposal for asparagine activation. Oligosaccharyltransferases catalyse the transfer of lipid-anchored glycans onto acceptor asparagine residues in substrate proteins. By assaying chemically modified peptide substrate analogues, Lizak et al . rule out all but one of the currently postulated catalytic mechanisms for this enzyme.