title: Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis.
abstract: 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.
authors: Department of Biology, Institute of Molecular Biology and Biophysics, Zurich, Switzerland.
authors: Sabina
authors: S
authors: Gerber
authors: Christian
authors: C
authors: Lizak
authors: Gaëlle
authors: G
authors: Michaud
authors: Monika
authors: M
authors: Bucher
authors: Tamis
authors: T
authors: Darbre
authors: Markus
authors: M
authors: Aebi
authors: Jean-Louis
authors: JL
authors: Reymond
authors: Kaspar P
authors: KP
authors: Locher
mesh_terms: D000595:Amino Acid Sequence; D044863:Campylobacter lari / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D007477:Ions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
chemical_list: D005452:Fluoresceins; D007477:Ions; D008565:Membrane Proteins; D010455:Peptides; C045132:4-carboxyfluorescein; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
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