ENZYMOLOGY OF ANTIBIOTIC RESISTANCE
Principal Investigator: R N Armstrong
Abstract: 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. Understanding the mechanisms of antimicrobial resistance is crucial for effective patient care in the clinic and essential for developing strategies to enhance biodefence against intentionally disseminated of pathogens. 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. 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, Listeria monocytogenes and Clostridium botulinum. Genomic and biochemical analysis from this project suggest that there are three distinct subgroups ofmetalloenzymes, 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 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 from Pseudomonas aeruginosa and its relatives FosB and FosX will be determined by X-ray crystallography. The chemical and ldnede mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+ in FosA and FosX by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB, and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. Potential transition state inhibitors will investigated by structural, spectroscopic and kinetic techniques. The thermodynamics of the interaction of substrates and inhibitors with the enzymes will be examined by isothermal titration calorimetry Particular emphasis will be placed on the enzymes from the pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum. The intent of this 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.
Funding Period: 1998-02-01 - 2009-07-15
more information: NIH RePORT
- Enzyme control of small-molecule coordination in FosA as revealed by 31P pulsed ENDOR and ESE-EPRCharles J Walsby
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Il 60208 3113, USA
J Am Chem Soc 127:8310-9. 2005....
- A model for glutathione binding and activation in the fosfomycin resistance protein, FosARachel E Rigsby
Department of Chemistry and the Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA
Arch Biochem Biophys 464:277-83. 2007..In the absence of co-crystal structural data with the thiol substrate, these results provide important insights into the role of GSH in catalysis...
- Structure and mechanism of the genomically encoded fosfomycin resistance protein, FosX, from Listeria monocytogenesKerry L Fillgrove
Department of Biochemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232 0146, USA
Biochemistry 46:8110-20. 2007..Kinetic analysis of mutants of active site residue E44 is consistent with its proposed roll as a general base catalyst in the addition of water to the antibiotic...
- Evolution of the antibiotic resistance protein, FosA, is linked to a catalytically promiscuous progenitorDaniel W Brown
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 0146, USA
Biochemistry 48:1847-9. 2009....