Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates

Han, Weiqing; Wu, Baolin; Li, Lei; Zhao, Guochang; Woodward, Robert L.; Pettit, Nicholas; Cai, Li; Thon, Vireak; Wang, Peng George

doi:10.1074/jbc.m111.308486

Cited by 67 publications

(65 citation statements)

References 38 publications

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“…3B). This relaxed specificity toward the lipid moiety of the glycan donor displayed by PglL has also been reported for the enzyme WaaL (22). The conserved functional and mechanistic features between WaaL ligases and O-OTases strengthen the idea of a tightly evolutionary relationship between these two families of enzymes, which basically catalyze the same reaction but toward different acceptors.…”

Section: Discussionmentioning

confidence: 56%

“…3A) in the reaction catalyzed by PglL. A similar strategy was recently employed to study WaaL glycan-donor specificity in vitro by Han et al (22). The glycosylation reaction was followed via quantification of the relative intensity of the signals of the bands corresponding to glycosylated and unglycosylated pilin.…”

Section: Pgll Transferase Activity Is Independent Of Divalent Cations-mentioning

confidence: 99%

“…The synthesis of the different synthetic organic compounds was performed following published methods (22)(23)(24), uridine diphosphate NЈ-diacetylbacillosamine (UDP-diNAcBac) was kindly provided by Martin Young (Institute for Biological Sciences, Ottawa, Ontario, Canada).…”

Section: Synthesis Of E Coli O Antigen O86 Linked To Different Lipidmentioning

confidence: 99%

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In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

Musumeci

Hug

Scott

et al. 2013

Journal of Biological Chemistry

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Background: PglL-like enzymes catalyze protein O-linked glycosylation in many bacteria. Results: The catalytic efficiency of this enzyme correlates with the length and conformation of the acyl chain of glycan donors. PglL also catalyzed glycan transfer from UDP-activated sugar. Conclusion: Lipid carriers are not essential but influence glycosylation. Significance: This work provides insights into the functionality of PglL.

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Section: Discussionmentioning

confidence: 56%

Section: Pgll Transferase Activity Is Independent Of Divalent Cations-mentioning

confidence: 99%

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In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

Musumeci

Hug

Scott

et al. 2013

Journal of Biological Chemistry

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“…Individual units are transported across this membrane and assembly of the OPS is catalyzed in the periplasm by the polymerase (7). The O chain is subsequently ligated to lipid A-core by the OPS ligase (8). In this pathway, chain-length modalities are conferred by the action of the polysaccharide copolymerase protein, Wzz.…”

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confidence: 99%

Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry inEscherichia coliO9a

King

Berry

Clarke

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The lengths of bacterial polysaccharides can be critical for their biological function. Unlike DNA or protein synthesis, where polymer length is implicit in the nucleic acid template, the molecular mechanisms for regulating polysaccharide length are poorly understood. Two models are commonly cited: a "molecular clock" regulates length by controlling the duration of the polymer extension process, whereas a "molecular ruler" determines length by measurement against a physical structure in the biosynthetic complex. Escherichia coli O9a is a prototype for the biosynthesis of O polysaccharides by ATP-binding cassette transporter-dependent processes. The length of the O9a polysaccharide is determined by two proteins: an extension enzyme, WbdA, and a termination enzyme, WbdD. WbdD is known to self-oligomerize and also to interact with WbdA. Changing either enzyme's concentration can alter the polysaccharide length. We quantified the O9a polysaccharide length distribution and the enzyme concentration dependence in vivo, then made mathematical models to predict the polymer length distributions resulting from hypothetical length-regulation mechanisms. Our data show qualitative features that cannot be explained by either a molecular clock or a molecular ruler model. Therefore, we propose a "variable geometry" model, in which a postulated biosynthetic WbdA-WbdD complex assembles with variable stoichiometry dependent on relative enzyme concentration. Each stoichiometry produces polymers with a distinct, geometrically determined, modal length. This model reproduces the enzyme concentration dependence and modality of the observed polysaccharide length distributions. Our work highlights limitations of previous models and provides new insight into the mechanisms of length control in polysaccharide biosynthesis.chain length | mathematical modeling | template-independent polymerization W hereas nucleic acids and proteins are synthesized from a template of specified length, carbohydrates are constructed template-free, implying that the mechanisms for length control must lie in the biosynthetic machinery itself.Lipopolysaccharide (LPS) is a glycolipid which constitutes the major and characteristic component of the outer leaflet of the outer membrane of most Gram negative bacterial cell envelopes (reviewed in ref. 1). Most bacteria produce LPS molecules that contain a conserved lipid A moiety linked to a short (core) oligosaccharide. In many species, a proportion of LPS molecules have an additional domain known as O polysaccharide (OPS), O antigen or O chain. OPS is a carbohydrate polymer in which the repeating unit (O unit) consists of a short oligosaccharide or sometimes a single sugar. OPS is synthesized template-free by glycosyltransferases and is found in lengths that vary from one repeat unit to several hundred sugars.The biological roles of OPS vary from species to species, but for bacterial pathogens, it commonly confers resistance to the bactericidal effects of host complement. The mechanisms of resistance can vary, so that ...

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“…Figure 1 shows that dLPS was as efficient as LPS in interfering with biofilm formation, which indicates the lipid A of LPS does not play a role in inhibiting biofilm formation by V. vulnificus. To verify this conclusion, a mutant V. vulnificus, which is not able to form O-Ag-substituted LPS due to a defect in incorporating O-Ag into the lipid A-core in the outer membrane, was constructed by deleting the rfaL gene encoding WaaL ligase (Han et al 2012). The LPS fraction extracted from the ΔrfaL/ hydrophilic.…”

Section: Vulnificus Biofilm Formation Was Inhibited By the Exogenomentioning

confidence: 95%

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

Lee

Hwang

et al. 2016

Biofouling

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The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.

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Defining Function of Lipopolysaccharide O-antigen Ligase WaaL Using Chemoenzymatically Synthesized Substrates

Cited by 67 publications

References 38 publications

In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry inEscherichia coliO9a

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

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