The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors

Abstract

Fosfomycin is a broad-spectrum antibiotic that has been underused due to the nature of the resistance mounted against it by various microorganisms. The work presented here elucidates the structure function relationship between the resistance enzyme, FosA, from Pseudomonas aeruginosa, and its substrate, glutathione. The relationship between enzyme and substrate is postulated by computational modeling and verified by mutagenesis and kinetic studies. The evolutionary relationship between the P. aeruginosa FosA enzyme and a structurally related but functionally divergent enzyme, the FosX enzyme from Mesorhizobium loti, is then explored using rational mutagenesis and DNA shuffling techniques as well as x-ray crystallography. Finally, the development and use of a high-throughput screen for the discovery of bioactive inhibitors of the P. aeruginosa FosA is described. Using a purified enzyme assay, several new chemical scaffolds have been discovered that effectively inhibit the FosA resistance enzyme. Two of these compounds show activity suggesting bioactivity and specificity.