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A catalytically essential motif in the external loop 5 of the bacterial oligosaccharyltransferase PglB

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Lizak C., Gerber S., Zinne C., Michaud Gaëlle, Schubert M., Chen F., Bucher M., Darbre Tamis, Zenobi R., Reymond Jean-Louis, Locher K.P.,
Project Exploring Peptide Topologies in Search for New Drugs
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Original article (peer-reviewed)

Journal The Journal of Biological Chemistry
Page(s) 735 - 746
Title of proceedings The Journal of Biological Chemistry
DOI 10.1074/jbc.m113.524751

Open Access

URL http://www.jbc.org/content/289/2/735.full.pdf+html
Type of Open Access Publisher (Gold Open Access)

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.
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