Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica.
WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaP(CT) domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be α-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.
Patel
Kinnari B
KB
Department of Microbiology and Immunology, Infectious Diseases Research Group, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6A5C1, Canada.
Furlong
Sarah E
SE
Valvano
Miguel A
MA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019081:O Antigens / Q000096:biosynthesis*; D017510:Protein Folding; D019779:Salmonella enterica / Q000276:immunology*
D001426:Bacterial Proteins; D019081:O Antigens