The O-acetylation of peptidoglycan is known to occur in a large number of bacteria including many important human pathogens, such as Staphylococcus aureus, species of Enterococcus, Bacillus anthracis, Helicobacter pylori, Campylobacter jejuni, and Neisseria gonnorrhoeae. This modification to the C-6 position of N-acetylmuramoyl residues of peptidoglycan inhibits the action of muramidases (lysozymes) of innate immune systems in a concentration dependant manner and it totally precludes the activity of the lytic transglycosylases, ubiquitous bacterial enzymes that are involved with the insertion of flagella, pili, and secretion/transport systems, as well as the general biosynthesis and turnover of the cell wall material. A two-component system for the O-acetylation of peptidoglycan was recently discovered in Gram-negative bacteria. An integral membrane protein, peptidoglycan O-acetyltransferase (Pat) A, is proposed to translocate acetate from cytoplasmic pools of acetyl-CoA through the cytoplasmic membrane to the outside of the cell for its transfer to peptidoglycan by extracellular PatB. In Gram-positive bacteria, such as S. aureus, a single protein, O-acetyltransferase (Oat), appears to be a fusion of PatA and PatB to catalyze both the translocation and transfer of acetate for peptidoglycan O-acetylation. We recently developed the first in vitro assay for any peptidoglycan O-acetyltransferase. Here, we apply the assay to the characterization of PatB from both Neisseria gonorrhoeae and Bacillus cereus, as well as the catalytic module of OatA from B. cereus. Thus, we present the first biochemical data on the specificity, pH-activity profile, Michaelis-Menten parameters, and mechanism of action of these O-acetyltransferases which involves a triad of Asp, His and Ser residues that appear to function in a manner analogous to serine esterases.
