Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

Liston, Sean D.; Clarke, Bradley R.; Greenfield, Laura K.; Richards, Michele R.; Lowary, Todd L.; Whitfield, Chris

doi:10.1074/jbc.m114.622480

Cited by 19 publications

(16 citation statements)

References 52 publications

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“…Several cytoplasmic membrane proteins implicated in the assembly of cell surface polysaccharides form oligomeric structures (Collins et al, 2017;Liston et al, 2015;Whitfield and Trent, 2014). Further, the MraY protein that initiates cell wall peptidoglycan synthesis forms dimers both in detergent micelles and in the membrane (Chung et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties

Ruan

Feria

Hamad

et al. 2018

Molecular Microbiology

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WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide, a conserved step in lipopolysaccharide biosynthesis. Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. Establishing the membrane topology of the WaaL protein family will enable understanding its mechanism and exploit it as a potential antimicrobial target. Applying oxidative labeling of native methionine/cysteine residues, we previously validated a topological model for Escherichia coli WaaL, which differs substantially from the reported topology of the Pseudomonas aeruginosa WaaL, derived from the analysis of truncated protein reporter fusions. Here, we examined the topology of intact E. coli and P. aeruginosa WaaL proteins by labeling engineered cysteine residues with the membrane-impermeable sulfhydryl reagent polyethylene glycol maleimide (PEG-Mal). The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. Further, we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL and that E. coli WaaL may adopt a functional dimer conformation.

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Section: Discussionmentioning

confidence: 99%

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties

Ruan

Feria

Hamad

et al. 2018

Molecular Microbiology

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“…A comparable sequence of enzymatic steps has been described for WbdA O9a , the polymerizing mannosyltransferase assembling the E. coli O9a antigen (62). WbdA O9a consists of two GT-B domains, and, during the synthesis of the tetrameric repeating unit, each domain is responsible for two consecutive mannose transfers in either ␣-(132)-or ␣-(133)-linkage, respectively (63,64).…”

Section: Volume 290 • Number 40 • October 2 2015mentioning

confidence: 99%

The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

Litschko

Romano

Pinto

et al. 2015

Journal of Biological Chemistry

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Background:The trimeric repeating unit forming the capsular polysaccharide of Neisseria meningitidis serogroup L comprises glycosidic and phosphodiester bonds. Results: We identified the two-domain capsule polymerase CslB of N. meningitidis serogroup L to assemble this complex trimer with UDP-GlcNAc as substrate. Conclusion: CslB represents a unique capsule polymerase in group 2 capsule expressing bacteria. Significance: Understanding capsule biosynthesis is mandatory for combatting encapsulated bacterial pathogens.

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“…The 1 H and 13 C NMR chemical shifts were assigned using a set of 2D experiments, including 1 H, 1 H COSY, total correlation spectroscopy (TOCSY), rotating-frame nuclear Overhauser effect correlation spectroscopy (ROESY), 1 H, 13 C heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) ( Table S1). The anomeric region of the 1 H NMR spectrum contained four signals of different intensities in the region δ 4.77-4.85, and a broad, low intensity signal at δ 4.46 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, the system includes a mechanism for chain-length determination, which dictates when OPS chains reach the correct length for termination to take place. This is established by structural features of a membrane-associated complex composed of WbdA and WbdD (13,16). Stoichiometry and geometry of the complex influences chain length control (5) but the distance between the polymerase and terminator catalytic sites provides the critical determinant of the OPS chain length.…”

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confidence: 99%

“…The polymannose OPSs shared by E. coli O8/O9/O9a and their K. pneumoniae O5/O3 counterparts offer the prototypes for the substrate-specific assembly pathway. A serotype-specific polymerase enzyme (designated WbdA), possessing two or more GDP-mannose-dependent GT catalytic sites, is responsible for building the repeat-unit structure of the Und-PP-linked polymannose OPS (12,13). The polymerase activity is opposed by a chain terminator (WbdD) that adds a methyl or phosphomethyl residue (depending on the serotype) to the nonreducing ends, creating completed OPS chains with optimal lengths within a limited (modal) size range before the involvement of the ABC transporter (14,15).…”

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confidence: 99%

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Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

Williams

Ovchinnikova

Koizumi

et al. 2017

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

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Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-β-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a β-linked Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering.microbial glycobiology | lipopolysaccharides | glycosyltransferases | glycan biosynthesis | molecular ruler B acterial surfaces possess an array of complex glycoconjugates (sugar-containing macromolecules) that play varied and vital roles in the biology of these organisms. In pathogens, glycoconjugates participate in adhesion, biofilm formation, and interaction with innate and adaptive immune responses. For example, in gram-negative bacteria such as Escherichia coli, long glycan chains in capsular polysaccharides and the O-antigen polysaccharide (OPS) components of lipopolysaccharide (LPS) molecules typically confer resistance to opsonophagocytosis and complement-mediated killing (1, 2). Effective protection depends on the amount and surface distribution of glycan, as well as glycan chain lengths suited for a given purpose. As an example, shorter OPS chains offer no resistance to complementmediated killing, whereas chains longer than an optimal size potentially represent an unnecessary energy cost while offering no additional advantage (3, 4). Despite its fundamental importance in colonization and virulence of bacterial pathogens, the molecular mechanisms of glycan chain-length regulation are often poorly understood (5). However, OPS biosynthesis provides influential prototypes for understanding the guiding principles underpinning chainlength regulatory mechanisms in bacterial glycoconjugates in general.The LPS glycolipid is a major component of the outer membrane of gram-negative bac...

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Domain Interactions Control Complex Formation and Polymerase Specificity in the Biosynthesis of the Escherichia coli O9a Antigen

Cited by 19 publications

References 52 publications

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O‐antigen ligases share similar membrane topology and biochemical properties

The Capsule Polymerase CslB of Neisseria meningitidis Serogroup L Catalyzes the Synthesis of a Complex Trimeric Repeating Unit Comprising Glycosidic and Phosphodiester Linkages

Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

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