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

Lizak, Christian; Gerber, Sabina; Zinne, Daria; Michaud, Gaëlle; Schubert, Mario; Chen, Fan; Bucher, Monika; Darbre, Tamis; Zenobi, Renato; Reymond, Jean‐Louis; Locher, Kaspar P.

doi:10.1074/jbc.m113.524751

Cited by 29 publications

(31 citation statements)

References 44 publications

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“…It is important to note that reductions of the in vivo output of the biosynthetic glycosylation pathway as a consequence of a PglK mutation may not directly correlate with the reduction of in vitro flipping rates because the rate-limiting step of the in vivo process is unknown. A similar phenomenon was previously observed for mutants of PglB that exhibited reductions of in vitro glycosylation rates by two orders of magnitude or more, but showing only marginally lower in vivo output 21,26,30,31 . We speculated that the hydrophobic grooves formed near the EH (Fig.…”

Section: Role Of External Helix Eh In Pglk-llo Interactionsupporting

confidence: 73%

“…By contrast, we exploited the fact that PglK can process LLOs with shortened oligosaccharides both in vivo and in vitro 20,25,26 . We therefore generated proteoliposomes by co-reconstituting PglK and a trisaccharide LLO (tLLO), which contained a reducing-end bacillosamine and two N-acetylgalactosamine (GalNAc) moieties ( Fig.…”

Section: Pglk In Vitro Llo Flipping and Atpase Activitymentioning

confidence: 98%

“…9a). The conversion of tLLO to hexa-saccharide LLO was confirmed by transferring the resulting oligosaccharides to fluorescently labelled acceptor peptides using purified oligosaccharyltransferase PglB, followed by SDS-PAGE analysis 26,30,31 . PglK-catalysed in vitro tLLO flipping was strictly dependent on ATP hydrolysis.…”

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confidence: 99%

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Structure and mechanism of an active lipid-linked oligosaccharide flippase

Pérez

Gerber

Boilevin

et al. 2015

Nature

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The flipping of membrane-embedded lipids containing large, polar head groups is slow and energetically unfavourable, and is therefore catalysed by flippases, the mechanisms of which are unknown. A prominent example of a flipping reaction is the translocation of lipid-linked oligosaccharides that serve as donors in N-linked protein glycosylation. In Campylobacter jejuni, this process is catalysed by the ABC transporter PglK. Here we present a mechanism of PglK-catalysed lipid-linked oligosaccharide flipping based on crystal structures in distinct states, a newly devised in vitro flipping assay, and in vivo studies. PglK can adopt inward- and outward-facing conformations in vitro, but only outward-facing states are required for flipping. While the pyrophosphate-oligosaccharide head group of lipid-linked oligosaccharides enters the translocation cavity and interacts with positively charged side chains, the lipidic polyprenyl tail binds and activates the transporter but remains exposed to the lipid bilayer during the reaction. The proposed mechanism is distinct from the classical alternating-access model applied to other transporters.

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Section: Role Of External Helix Eh In Pglk-llo Interactionsupporting

confidence: 73%

Section: Pglk In Vitro Llo Flipping and Atpase Activitymentioning

confidence: 98%

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Structure and mechanism of an active lipid-linked oligosaccharide flippase

Pérez

Gerber

Boilevin

et al. 2015

Nature

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190

215

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Section: Gt Foldsmentioning

confidence: 99%

“…Two structures are now known, and each contains an N-terminal TM region and a C-terminal GT domain, which contains a DxD motif (Gloster, 2014). Utilizing a lipid-phosphate-linked sugar donor, the GT-C folds appear to require divalent cations for activity (Matsumoto et al, 2013;Lizak et al, 2014).GTs play a number of important biological roles. The canonical mechanism involves connecting the hydroxyls of two sugars through a glycosidic bond; however, the acceptor can actually comprise a large number of functional groups including nitrogen, sulfur, and carbon (Lairson et al, 2008).…”

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confidence: 99%

Structural Studies of Biopolymer Membrane Transport

Morgan¹

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The selective transport of specific substrates across the membrane is an essential part of biology.Organisms have evolved a basic mechanism for the transport of ions and small molecule such as sugars across the membrane. This mechanism is termed 'alternating access' and involves the alternating exposure of a substrate-binding site to either side of the membrane. While the molecular details of 'alternating access' have been revealed for a number of different transporters, this scheme seems infeasible for the transport of long biopolymers such as nucleic acids, proteins, and polysaccharides because 'alternating access' requires that the transporter forms a binding site that is large enough to bind the entire substrate. Here, I present novel data addressing the transport mechanism for two bacterial biopolymer transporter proteins: Bacterial Cellulose Synthase which synthesizes and secretes the polysaccharide, cellulose; and PrtD, an ABC transporter which is involved in the secretion of an extracellular protease.Cellulose is a linear polymer of glucose units that forms a major component of plant cell walls as well as many bacterial biofilms. In bacteria, the components required for cellulose biosynthesis are encoded in a single operon minimally made up of the genes bcsA, bcsB, bcsC, and bcsZ. BcsA is an integral inner-membrane (IM) glycosyltransferase enzyme that is responsible for coupling the synthesis of cellulose with its transport across the IM. BcsB is a periplasmic protein with Cterminal IM anchor, and BcsB forms a complex with BcsA (BcsA-B) that is sufficient for in vitro cellulose synthesis. BcsZ is a periplasmic cellulase enzyme, and BcsC is an outer-membrane β-barrel that presumably forms a pore for the cellulose polymer to cross the outer membrane.iii BcsA-B activity is stimulated by the bacterial signaling molecule, cyclic-di-GMP (c-di-GMP), which is a key regulator of biofilm formation. I present crystal structures of the c-di-GMP-bound BcsA-B complex in the presence and absence of UDP, a competitive inhibitor and substrate mimic. The structures reveal that c-di-GMP releases an auto-inhibited state of the enzyme. A salt bridge stabilizes one of the signature c-di-GMP-binding Arg residues in a position to tether a conserved 'gating loop' in front of the active site. The binding of c-di-GMP releases the tether and allows substrate to access the active site. Additionally, the UDP-bound structure reveals an additional role for the 'gating loop' in coordinating substrate at the active site. Functional experiments confirm the structural interpretation by revealing that disruption of the auto-inhibitory salt bridge by mutagenesis generates a constitutively-active cellulose synthase. The mechanistic insights presented here represent the first examples of how c-di-GMP allosterically modulates enzymatic functions.To address the mechanism by which BcsA-B transports cellulose across the IM, I use in crystallo enzymology with the c-di-GMP-bound BcsA-B crystals. Because crystallized BcsA-B is catalytically a...

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