Investigation of Protein and Inhibitor Binding to O-Acetylpeptidoglycan Esterase 1 from Neisseria gonorrhoeae

Mark Ecclestone, B.Sc. (Hons.) Advisor: Dr. A. Clarke O-Acetylpeptidoglycan esterase 1 (Ape1) is a periplasmic esterase present in pathogenic organisms that produce O-acetylated peptidoglycan. Ape1a plays a crucial role in the growth of these bacteria by regulating the turnover of peptidoglycan through catalytic removal of the C-6 acetyl group of the modified peptidoglycan. Specifically, Ape1a activity is necessary for peptidoglycan turnover by the main autolytic enzymes, lytic transglycosylases (LTs); LTs are blocked by the presence of an acetyl group on the C-6 hydroxyl of N-acetylmuramic acid. Ape1 is thought to be bound to an LT as part of a multi-enzyme complex responsible for cell division. Purpurin has been observed to inhibit the esterase activity of Ape1a in vitro and it has been shown to inhibit the growth of pathogenic bacteria that produce O-acetylpeptidoglycan. Membrane-perturbing compounds, such as EDTA and aminoglycosides, have been found to aid purpurin with inhibiting the growth of Gram-negative bacteria. Work presented in this thesis investigated i) the potential binding partners of Ape1 from N. gonorrhoeae, ii) the inhibitory mechanism of purpurin on Ape1, and iii) the antibacterial mechanism of purpurin with membrane-perturbing agents. This work found that Ape1 binds to FtsZ in a presumed multi-enzyme complex. The 2-hydroxyl group of anthraquinones, such as purpurin or alizarin, needs to be deprotonated to work as a suitable inhibitor of Ape1. Purpurin was determined to work synergistically with aminoglycosides against Bacillus cereus species, resulting in an extended lag time and elongated cells. Heightened O-acetylation levels and lack of septal formation suggest an effect on the division machinery.