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

Christian Lizak, Sabina Gerber, Gaëlle Michaud, Mario Schubert, Yao-Yun Fan, Monika Bucher, Tamis Darbre, Markus Aebi, Jean-Louis Reymond and Kaspar P. Locher ()
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Christian Lizak: Institute of Molecular Biology and Biophysics, ETH Zürich
Sabina Gerber: Institute of Molecular Biology and Biophysics, ETH Zürich
Gaëlle Michaud: University of Berne
Mario Schubert: Institute of Molecular Biology and Biophysics, ETH Zürich
Yao-Yun Fan: Institute of Microbiology, ETH Zürich
Monika Bucher: Institute of Molecular Biology and Biophysics, ETH Zürich
Tamis Darbre: University of Berne
Markus Aebi: Institute of Microbiology, ETH Zürich
Jean-Louis Reymond: University of Berne
Kaspar P. Locher: Institute of Molecular Biology and Biophysics, ETH Zürich

Nature Communications, 2013, vol. 4, issue 1, 1-11

Abstract: Abstract 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.

Date: 2013
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DOI: 10.1038/ncomms3627

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