title: The C-terminal domain of the Salmonella enterica WbaP (UDP-galactose:Und-P galactose-1-phosphate transferase) is sufficient for catalytic activity and specificity for undecaprenyl monophosphate.
abstract: Two families of membrane enzymes catalyze the initiation of the synthesis of O-antigen lipopolysaccharide. The Salmonella enterica Typhimurium WbaP is a prototypic member of one of these families. We report here the purification and biochemical characterization of the WbaP C-terminal (WbaP(CT)) domain harboring one putative transmembrane helix and a large cytoplasmic tail. An N-terminal thioredoxin fusion greatly improved solubility and stability of WbaP(CT) allowing us to obtain highly purified protein. We demonstrate that WbaP(CT) is sufficient to catalyze the in vitro transfer of galactose (Gal)-1-phosphate from uridine monophosphate (UDP)-Gal to the lipid carrier undecaprenyl monophosphate (Und-P). We optimized the in vitro assay to determine steady-state kinetic parameters with the substrates UDP-Gal and Und-P. Using various purified polyisoprenyl phosphates of increasing length and variable saturation of the isoprene units, we also demonstrate that the purified enzyme functions highly efficiently with Und-P, suggesting that the WbaP(CT) domain contains all the essential motifs to catalyze the synthesis of the Und-P-P-Gal molecule that primes the biosynthesis of bacterial surface glycans.
authors.affiliation: Department of Microbiology and Immunology, Center for Human Immunology, University of Western Ontario,London, ON, Canada N6A 5C1.
authors.forename: Kinnari B
authors.initials: KB
authors.lastname: Patel
authors.forename: Ewa
authors.initials: E
authors.lastname: Ciepichal
authors.forename: Ewa
authors.initials: E
authors.lastname: Swiezewska
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Valvano
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification; D011189:Potassium Chloride / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology*; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014531:Uridine Diphosphate Galactose; D012965:Sodium Chloride; D011189:Potassium Chloride; D010770:Phosphotransferases