title: 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.
authors: Centre for Human Immunology, Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada.
authors: Xiang
authors: X
authors: Ruan
authors: David E
authors: DE
authors: Loyola
authors: Cristina L
authors: CL
authors: Marolda
authors: José M
authors: JM
authors: Perez-Donoso
authors: Miguel A
authors: MA
authors: Valvano
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism; D007477:Ions / Q000737:chemistry / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019081:O Antigens / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D019081:O Antigens; D000255:Adenosine Triphosphate; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D008025:Ligases; D019729:Carbon-Oxygen Ligases; C528973:WaaL protein, Pseudomonas aeruginosa; D008274:Magnesium