title: Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation.
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.
authors.affiliation: 1] Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland [2].
authors.forename: Christian
authors.initials: C
authors.lastname: Lizak
authors.forename: Sabina
authors.initials: S
authors.lastname: Gerber
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Michaud
authors.forename: Mario
authors.initials: M
authors.lastname: Schubert
authors.forename: Yao-Yun
authors.initials: YY
authors.lastname: Fan
authors.forename: Monika
authors.initials: M
authors.lastname: Bucher
authors.forename: Tamis
authors.initials: T
authors.lastname: Darbre
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Reymond
authors.forename: Kaspar P
authors.initials: KP
authors.lastname: Locher
mesh_terms: D000577:Amides / Q000378:metabolism*; D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D016123:Campylobacter jejuni / Q000201:enzymology / Q000235:genetics; D044863:Campylobacter lari / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005973:Glutamine / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006714:Homoserine / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D005973:Glutamine; D001224:Aspartic Acid; D006714:Homoserine; D001216:Asparagine; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase