Characterization of Erythromycin Esterases: A Genomic Enzymology Approach to Macrolide Resistance

Abstract

<p>Enzymatic inactivation of antibiotics is a successful strategy employed by antibiotic resistant clinical pathogens. Genes coding for the antibiotic inactivating enzyme erythromycin esterase, or Ere, have been found in a variety of bacterial species, from clinical isolates to environmental soil-dwelling organisms. In order to better understand this family of proteins and this mode of macrolide resistance, four erythromycin esterases (EreA from Providencia stuartii, EreB from E. coli, and two putative Ere's from Saccharopolyspora erythraea and Bacillus cereus) were purified and characterized using a genomic enzymology approach. A robust quantitative enzyme assay was developed, and kinetic parameters for these different enzymes were compared. In the absence of a crystal structure for EreA or EreB, a model for EreB was developed based on the existing structure of the putative esterase from B. cereus. The importance of some conserved and potentially catalytic residues was examined using site-directed mutagenesis. Combined with mutagenesis, inhibitors, pH studies and solvent isotope effects were used to investigate potential enzyme mechanisms and two potential mechanisms were proposed for this family of proteins. A greater understanding of the function and mechanisms of antibiotic resistance elements such as the erythromycin esterases may provide valuable insights to aid in the ongoing struggle against resistant organisms in clinical settings.</p>