A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB

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Author(s) Lizak, Christian, Gerber, Sabina, Zinne, Daria, Michaud, Gaëlle, Schubert, Mario, Chen, Fan, Bucher, Monika, Darbre, Tamis, Zenobi, Renato, Reymond, Jean-Louis, Locher, Kaspar P.
Publication Type Journal Items, Publication Status: Published
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Title A Catalytically Essential Motif in External Loop 5 of the Bacterial Oligosaccharyltransferase PglB
Author(s) Lizak, Christian
Gerber, Sabina
Zinne, Daria
Michaud, Gaëlle
Schubert, Mario
Chen, Fan
Bucher, Monika
Darbre, Tamis
Zenobi, Renato
Reymond, Jean-Louis
Locher, Kaspar P.
Journal or Series Title The journal of biological chemistry
Volume Number 289
Issue Number 2
Start Page 735
End Page 746
ISSN 0021-9258
1083-351X
Publisher The American Society for Biochemistry and Molecular Biology
Publication Place Bethesda, MD
Publication Date 2014-01
Keyword(s) Campylobacter
Cysteine-mediated Cross-linking
Enzyme Catalysis
Enzyme Kinetics
Enzyme Mechanisms
Glycoprotein Biosynthesis
Glycosyltransferases
Membrane Enzymes
Oligosaccharide
Protein Structure
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 (Tyr(293)) 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.
DOI 10.1074/jbc.M113.524751
Additional Notes Received 4 October 2013, Revision received 5 November 2013
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03652
03430
Organisational Unit(s)
NEBIS System Number 000040237
Source Database ID WOS-000330541200013
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@article{Lzk2014,
  author = "Lizak, Christian and Gerber, Sabina and Zinne, Daria and Michaud, Ga{\"{e}}lle and Schubert, Mario and Chen, Fan and Bucher, Monika and Darbre, Tamis and Zenobi, Renato and Reymond, Jean-Louis and Locher, Kaspar P.",
  title = "{A} {C}atalytically {E}ssential {M}otif in {E}xternal {L}oop 5 of the {B}acterial {O}ligosaccharyltransferase {P}gl{B}",
  journal = "The journal of biological chemistry",
  year = 2014,
  volume = "289",
  number = "2",
  pages = "735--746",
  month = jan,
}


E-Citations record created: Mon, 10 Mar 2014, 09:59:18 CET