Modification of the bacterial cell wall heteropolymer peptidoglycan by addition of an acetyl group to the C-6 hydroxyl group of N-acetylmuramoyl residues is known to inhibit the activity of muramidases (lysozymes) of innate immune systems. The O-acetylation of peptidoglycan also precludes the action of intrinsic lytic transglycosylases, enzymes that require a free C-6 hydroxyl group to generate their 1,6-anhydromuropeptide products. This class of autolysins is ubiquitous in peptidoglycan-synthesizing bacteria as they are responsible for insertion of pores and flagella, spore formation, and the general metabolism of peptidoglycan. We recently discovered a cluster of genes in the Neisseria gonorrhoeae chromosome that are proposed to participate in peptidoglycan O-acetylation (Weadge, J. T., Pfeffer, J. M., and Clarke, A. J. (2005) BMC Microb. 5, 49). In the current study, we demonstrate that one of these genes, ape1 functions as an O-acetylpeptidoglycan esterase. The ape1 gene was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag. The expressed protein was purified to apparent homogeneity and assayed for activity as an esterase using three different assays involving high-performance liquid chromatography and chromogenic detection methods which measured the release of ester-linked acetate from a variety of polymer and soluble substrates. These assays demonstrated that Ape1 has a higher specific activity on O-acetylated peptidoglycan compared to O-acetylated xylan. Consequently, Ape1 represents the first enzyme characterized as an O-acetylpeptidoglycan esterase. The physicochemical and kinetic parameters of Ape1 were determined using soluble chromogenic substrates for convenience. Thus, its pH optima for stability and activity were observed to be 6.0 and 6.2, respectively, while its optimum temperature for activity was 55 degrees C. Two forms of truncated Ape1 are generated in E. coli, one lacked the complete predicted N-terminal signal sequence, while the second involved a proteolytic cleavage within this signal sequence. The smaller truncated form was localized predominantly to the periplasm, whereas the larger form was mainly associated with the outer membrane, and to a lesser extent, the cytoplasmic membrane, sites expected for the maintenance of peptidoglycan.