Five decades ago the introduction modern antibiotics began a revolution in effective therapy for infectious diseases. Today those advances in chemotherapy are in jeopardy due to the increasingly common appearance of drug resistant strains of microorganisms. The treatment of increasing numbers of immunocompromised individuals represents at once a clinical challenge that must be met and a venue for the selection of new, more resistant strains. It is not clear that enterococci resistant to all major classes of antibiotics are an immediate threat in the clinic. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. Early in the last decade plasmid-mediated resistance to fosfomycin was observed in the clinic. Subsequent investigations established that the resistance plasmids encode metalloproteins (FosA or FosB) that catalyze the addition of glutathione to the antibiotic, rendering it inactive. More recently, chromosomal resistance genes encoding putative fosfomycin canoeists have been described. The objectives of this research project are to elucidate the catalytic mechanisms, and structures of enzymes involved in the resistance of microorganisms to fosfomycin. These objectives include, the construction of high-level expression systems for the proteins, the elucidation of their catalytic mechanisms, and the determination of their three-dimensional structures by X-ray crystallography. The investigations of the fosfomycin resistance proteins FosA and FosB will include: (i) a determination of the kinetic mechanism of catalysis by pre-steady state and steady state kinetic techniques; (ii) elucidation of chemical mechanism of catalysis including the role of the role of the metal ion in the reaction by magnetic resonance (EPR and ENDOR) techniques; (iii) the determination of the enzyme-substrate interactions important in catalysis; and (iv) determination of the three-dimensional structures of the apoenzyme and the holoenzyme with bound Fosfomycin.
The specific aims of the project with respect to the fosfomycin kinase FosC include: (i) synthesis if the gene and expression of the protein in E.coli; (ii) a determination of the structure of the product of the enzymatic reaction and an investigation of the kinetic mechanism of the enzyme including the divalent cation requirement; (iii) and examination of the hypothesis that FosC. and another kinase FomA are related and (iv) initiation of X-ray crystallographic investigations of the structure of FosC. It is anticipated that this investigation will establish the mechanistic and structural foundation for design of new drugs to counter resistance to fosfomycin. The project is a response to program announcement PA-97-026, Aspergillosis, Ehrlichioses and Drug Resistance.
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