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
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 (WbaPCT) domain harboring one putative
transmembrane helix and a large cytoplasmic tail. An Nterminal
thioredoxin fusion greatly improved solubility and
stability of WbaPCT allowing us to obtain highly purified
protein. We demonstrate that WbaPCT 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 theWbaPCT 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