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Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis.

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Gerber Sabina, Lizak Christian, Michaud Gaëlle, Bucher Monika, Darbre Tamis, Aebi Markus, Reymond Jean-Louis, Locher Kaspar P,
Project Exploring Peptide Topologies in Search for New Drugs
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Original article (peer-reviewed)

Journal The Journal of biological chemistry
Volume (Issue) 288(13)
Page(s) 8849 - 61
Title of proceedings The Journal of biological chemistry
DOI 10.1074/jbc.m112.445940


N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the -2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.