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Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium

The Gram‐positive pathogen Enterococcus faecium is one of the leading causes of hospital‐acquired vancomycin resistant enterococci (VRE) infections. E. faecium has extensive multidrug resistance and accounts for more than two million infections in the United States each year. FosB is a fosfomycin resistance enzyme found in Gram‐positive pathogens like E. faecium. Typically, the FosB enzymes are Mn2+‐dependent bacillithiol (BSH) transferases that inactivate fosfomycin through nucleophilic addition of the thiol to the antibiotic. However, our kinetic analysis of FosBEf shows that the enzyme does not utilize BSH as a thiol substrate, unlike the other well characterized FosB enzymes. Here we report that FosBEf is a Mn2+‐dependent L‐cys transferase. In addition, we have determined the three‐dimensional X‐ray crystal structure of FosBEf in complex with fosfomycin at a resolution of 2.0 Å. A sequence similarity network (SSN) was generated for the FosB family to investigate the unexpected substrate selectivity. Three non‐conserved residues were identified in the SSN that may contribute to the substrate selectivity differences in the family of enzymes. Our structural and functional characterization of FosBEf establishes the enzyme as a potential target and may prove useful for future structure‐based development of FosB inhibitors to increase the efficacy of fosfomycin.