EXCEEDTHE SPACE PROVIDED. In the last two decades it has become increasingly clear that the efficacy of antibiotics for the treatment of infectious diseases is in jeopardy due to the common appearance of drug resistant strains of microorganisms. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. A decade after its introduction plasmid-mediated resistance to fosfomycin was observed in the clinic. Investigations supported by this project have established that the resistance is due to a metalloenzyme (FosA) that catalyzes the addition ofglutathione to the antibiotic, rendering it inactive. Moreover, similar resistance elements have now been shown to exist in the genomes of several pathogenic microorganisms including, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus anthrasis, Brucella melitensis, and Listeria monocytogenes. Genomic and biochemical analysis suggest that there are three distinct subgroups of metalloenzymes, termed FosA, FosB and FosX, that confer resistance through somewhat different chemical mechanisms. The objectives of this research project are to identify plasmid and genomically encoded proteins involved in microbial resistance to the antibiotic fosfomycin and to elucidate the underlying structural and mechanistic enzymology of resistance. These objectives will be accomplished by integrating enzymological, biophysical and genomic analyses of the resistance problem. The three-dimensional structures of the FosA homologue PA 1129 from Pseudomonas aeruginosa and its close relatives will be determined X-ray crystallography and mass spectrometry. The chemical and kinetic mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+in FosA by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB and the inhibition of these enzymes by phosphonoformate with particular emphasis on the enzymes from the pathogens Pseudomonas aeruginosa and Staphylococcus aureus; and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. The thermodynamics and molecular dynamics of the interaction of metals, substrates and inhibitors with FosA and FosB will be examined by: (i) a determination of the thermodynamics of binding of metals, substrate and inhibitor ligands by isothermal titration calorimetry and; (ii) an NMR investigation of the changes in the molecular dynamics of the protein on metal and substrate binding to assess the contribution of high-frequency motions to the overall binding energetics. The intent of these investigation is to establish the mechanistic and structural bases for the design of drugs to counter both plasmid borne and genomically encoded resistance to fosfomycin. PERFORMANCE SITE ========================================Section End===========================================
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