Substrate Specificity and Kinetic Characterization of Peptidoglycan O -Acetyltransferase B from Neisseria gonorrhoeae

The O-acetylation of the essential cell wall polymer pepti- doglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococ- cus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neis- seria meningitidis. With Gram-negative bacteria, the transloca- tion of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidogly- can by O-acetyltransferase PatB, whereas a single bimodal mem- brane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro bio- chemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosac- charides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valu- able for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.