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Mechanism of Action of Neisseria gonorrhoeae O-Acetylpeptidoglycan Esterase, an SGNH Serine Esterase

Significance Statement

The O-acetylation of peptidoglycan (PG) is presently known to occur in greater than 50 distinct eubacterial species, including numerous human pathogens such as Bacillus anthracis, Campylobacter jejuni, Helicobacter pylori, Neisseria gonorrhoeae, N. meningitidis, Staphylococcus aureus, and all species of Enterococcus examined thus far.  This modification, which occurs at the C-6 hydroxyl of the N-acetylmuramoyl residues within the heteropolymer’s glycan backbone, not only serves as an autolytic regulatory mechanism during cell wall metabolism, but has also been demonstrated to contribute to pathogenesis and persistence within a host (1).  Not surprisingly, interruption of the PG O-acetylation pathway, either through inactivation of the encoding O-acetyltransferases or through inhibition of O-acetylpeptidoglycan esterase (Ape) has been found to significantly affect cell survivability in vivo by several research groups, including our own (2-6).  In light of the continued rapid development and subsequent global dissemination of resistance mechanisms to current antimicrobials, the enzymes involved in the pathway(s) responsible for the modification may thus represent potential targets for the development of novel therapeutic agents.  However, to facilitate this possibility, a greater understanding of their respective mechanism(s) is required.  Site-directed replacement coupled with kinetic characterization and binding studies of the resultant engineered variants was thus used in this study to further our understanding of Ape, an enzyme that is responsible for the removal of the modification, thereby permitting the continued maintenance and metabolism of the PG sacculus primarily in Gram-negative species.  In accordance with the proposed mechanism, Gly236 and Asn268 were identified as participating in the stabilization of the negatively charged tetrahedral transitional species through the formation of an oxyanion hole, while several other invariant residues are proposed to either appropriately position the catalytic residues and/or participate in substrate binding.  These findings combined with the recent advent of defined soluble substrate mimics for the enzyme, which in turn may serve as scaffolds for further modification (7), may thus aid in the development and design of specific inhibitory compounds.



1.         Moynihan, P.J., and Clarke, A.J. (2011) Int. J. Biochem. Cell Biol.  43, 1655-1659.

2.         Aubry, C. et al. (2011) J. Infect. Dis.  204, 731-740.

3.         Hebert, L. et al. (2007) Infect. Immun.  75, 5390-5398.

4.         Rae, C.S. et al. (2011) Infect. Immun.  79, 3596-3606.

5.         Shimada, T. et al. (2010) Cell Host Microbe  7, 38-49.

6.         Pfeffer, J.M., and Clarke, A.J., Identification of the First Known Inhibitors of O- Acetylpeptidoglycan Esterase: A Potential New Antibacterial Target (2012) ChemBioChem  13, 722-731.

7.         Hadi, T. et al., Water-Soluble Substrates of the Peptidoglycan-Modifying Enzyme O-Acetylpeptidoglycan Esterase (Ape1) from Neisseria gonorrheae (2011) J. Org. Chem.  76, 1118-1125.


Journal Reference

Pfeffer JM, Weadge JT, Clarke AJ.

J Biol Chem. 2013 Jan 25;288(4):2605-13.

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.


O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted {Alpha}/{Beta}-hydrolase fold containing a central twisted four-stranded parallel {Beta}-sheet flanked by six {Alpha}-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding.

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