title: The C-terminal Domain of the Escherichia coli WaaJ glycosyltransferase is important for catalytic activity and membrane association.
abstract: The waaJ gene encodes an alpha-1,2-glucosyltransferase involved in the synthesis of the outer core region of the lipopolysaccha-ride of some Escherichia coli and Salmonella isolates. WaaJ belongs to glycosyltransferase CAZy family 8, characterized by the GT-A fold, a DXD motif, and by retention of configuration at the anomeric carbon of the donor sugar. Detailed kinetic and structural information for bacterial family 8 glycosyltransferases has resulted from studies of Neisseria meningitidis LgtC. As many as 28 amino acids could be deleted from the C terminus of LgtC without affecting its in vitro catalytic behavior. This C-terminal domain has a high ratio of positively charged and hydrophobic residues, a feature conserved in WaaJ and some other family 8 representatives. Unexpectedly, deletion of as few as five residues from the C terminus of WaaJ resulted in substantially reduced in vivo activity. With deletions of 15 residues or less, activity was only detected when levels of expression were elevated. No in vivo activity was detected after the removal of 20 amino acids, regardless of expression levels. Longer deletions (20 residues and greater) compromised the ability of WaaJ to associate with the membrane. However, the reduced in vivo activity in enzymes lacking 5-12 C-terminal residues also reflected a dramatic drop in catalytic activity in vitro (a 294-fold decrease in the apparent kcat/Km,LPS). Deletions removing 20 or more residues resulted in a protein showing no detectable in vitro activity. Therefore, the C-terminal domain of WaaJ plays a critical role in enzyme function.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Leipold
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Kaniuk
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014166:Transferases / Q000737:chemistry / Q000235:genetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D014166:Transferases; D006602:Hexosyltransferases; C501543:waaU protein, E coli; C042308:phosphatidylglycerol glyceryl transferase