The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases*

Abstract

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5'-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X(9)GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the β-configuration, whereas the sugars bound to Und-PP are in the α-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

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

“…This result is in agreement with data obtained by Ruan et al (48) for the O antigen ligase WaaL from E. coli, which is homologous to PglL and catalyzes the glycan transfer from the undecaprenylpyrophosphate lipid carrier to lipid A. These authors showed that WaaL does not require a divalent metal cation for its activity (48).…”

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

“…The WaaL enzyme that ligates nascent O-antigens to lipid A is specific for Und-PP-linked glycan donors, and its mechanism and architecture are conserved across species (58). This enzyme is unaffected by the pathway of O-antigen biosynthesis.…”

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

“…The experiments of this work were carried out on a WaaL construct with a C-terminal FLAG-His10x tag, containing 32 additional residues, and resulting in an overall average MW of 50794.8 Da. The protein was purified from E. coli JM105v by Ni 2+ -affinity chromatography, and it was subsequently concentrated using a 30 kDa cutoff membrane as described [59]. This method afforded~1 mg of WaaL protein per liter of culture.…”

“…The experiments of this study were conducted on DM-solubilized protein. Enzyme activity was verified by an in vitro ligation assay [59]. Buffer exchange yielded stock solutions containing 100 μM WaaL, 0.05% (wt/vol) DM, 150 mM sodium chloride, 12.5 mM imidazole, 10% glycerol, and 50 mM sodium phosphate (pH 8).…”

“…The O antigen is synthesized as a lipid-linked glycan intermediate, and it is ligated to the lipid A-core oligosaccharide prior to export of the complete LPS molecule to the bacterial surface [58]. WaaL is an inner membrane IMP that catalyzes this ligation [13,15,59]. Application of the prediction algorithm TMHHM [60] in combination with biochemical assays yielded a topology model of E. coli WaaL that involves 12 transmembrane helices ( Figure 1 [13]).…”