1. The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases.
- Author
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Ruan X, Loyola DE, Marolda CL, Perez-Donoso JM, and Valvano MA
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins isolation & purification, Carbon-Oxygen Ligases chemistry, Carbon-Oxygen Ligases isolation & purification, Conserved Sequence, Escherichia coli chemistry, Escherichia coli metabolism, Glycosyltransferases chemistry, Glycosyltransferases isolation & purification, Hexosyltransferases chemistry, Hexosyltransferases metabolism, Ions chemistry, Ligases chemistry, Lipopolysaccharides chemistry, Magnesium chemistry, Membrane Proteins chemistry, Membrane Proteins metabolism, Molecular Sequence Data, O Antigens chemistry, Pseudomonas aeruginosa chemistry, Pseudomonas aeruginosa metabolism, Bacterial Proteins metabolism, Carbon-Oxygen Ligases metabolism, Glycosyltransferases metabolism, Ions metabolism, Ligases metabolism, Lipopolysaccharides metabolism, Magnesium metabolism, O Antigens metabolism
- 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.
- Published
- 2012
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