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

Author

Listed:
  • Christian Lizak

    (Institute of Molecular Biology and Biophysics, ETH Zürich)

  • Sabina Gerber

    (Institute of Molecular Biology and Biophysics, ETH Zürich
    Present address: GlycoVaxyn AG, Grabenstrasse 3, 8952 Schlieren, Switzerland)

  • 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)

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.

Suggested Citation

  • Christian Lizak & Sabina Gerber & Gaëlle Michaud & Mario Schubert & Yao-Yun Fan & Monika Bucher & Tamis Darbre & Markus Aebi & Jean-Louis Reymond & Kaspar P. Locher, 2013. "Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation," Nature Communications, Nature, vol. 4(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:4:y:2013:i:1:d:10.1038_ncomms3627
    DOI: 10.1038/ncomms3627
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    Cited by:

    1. Ana S. Ramírez & Mario Capitani & Giorgio Pesciullesi & Julia Kowal & Joël S. Bloch & Rossitza N. Irobalieva & Jean-Louis Reymond & Markus Aebi & Kaspar P. Locher, 2022. "Molecular basis for glycan recognition and reaction priming of eukaryotic oligosaccharyltransferase," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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