title: A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.
abstract: Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr293) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr293. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.
authors.affiliation: From the Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, Schafmattstrasse 20, 8093 Zurich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Lizak
authors.forename: Sabina
authors.initials: S
authors.lastname: Gerber
authors.forename: Daria
authors.initials: D
authors.lastname: Zinne
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Michaud
authors.forename: Mario
authors.initials: M
authors.lastname: Schubert
authors.forename: Fan
authors.initials: F
authors.lastname: Chen
authors.forename: Monika
authors.initials: M
authors.lastname: Bucher
authors.forename: Tamis
authors.initials: T
authors.lastname: Darbre
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Reymond
authors.forename: Kaspar P
authors.initials: KP
authors.lastname: Locher
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D044863:Campylobacter lari / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D010455:Peptides; C023023:lipid-linked oligosaccharides; D014443:Tyrosine; D001216:Asparagine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: Campylobacter; Cysteine-mediated Cross-linking; Enzyme Catalysis; Enzyme Kinetics; Enzyme Mechanisms; Glycoprotein Biosynthesis; Glycosyltransferases; Membrane Enzymes; Oligosaccharide; Protein Structure