Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli.
The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.
Amer
Amal O
AO
Departments of Microbiology and Immunology1 and Medicine2, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Valvano
Miguel A
MA
Departments of Microbiology and Immunology1 and Medicine2, University of Western Ontario, London, Ontario N6A 5C1, Canada.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D002412:Cations / Q000378:metabolism; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000096:biosynthesis*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*
D000942:Antigens, Bacterial; D002412:Cations; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D019081:O Antigens; D001224:Aspartic Acid; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli