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Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium
Vanessa Wiltsie1, Skye Travis1, Madeline R Shay1
1Department of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa, Alabama, USA.
Abstract:
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.
Insights
Enterococcus faecium causes hospital infections. Researchers found its FosB enzyme uses L-cysteine, not BSH, to resist fosfomycin, offering new targets for drug development.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Enterococcus faecium, a Gram-positive pathogen, is a primary cause of hospital-acquired vancomycin-resistant enterococci (VRE) infections.
- E. faecium exhibits extensive multidrug resistance, leading to over two million infections annually in the US.
- FosB is a key fosfomycin resistance enzyme in Gram-positive pathogens, typically acting as a manganese-dependent bacillithiol transferase.
Purpose of the Study:
- To investigate the substrate specificity and mechanism of the FosB enzyme from E. faecium (FosB Ef).
- To determine the three-dimensional structure of FosB Ef in complex with fosfomycin.
- To identify structural determinants responsible for altered substrate selectivity within the FosB enzyme family.
Main Methods:
- Kinetic analysis to determine enzyme substrate utilization.
- X-ray crystallography to resolve the 3D structure of FosB Ef-fosfomycin complex.
- Sequence similarity network (SSN) analysis of the FosB family.
Main Results:
- FosB Ef functions as a manganese-dependent L-cysteine transferase, differing from other characterized FosB enzymes that use bacillithiol.
- The X-ray crystal structure of FosB Ef complexed with fosfomycin was determined at 2.0 kA resolution.
- SSN analysis identified three non-conserved residues potentially responsible for the divergent substrate selectivity in the FosB family.
Conclusions:
- FosB Ef exhibits unique substrate selectivity, utilizing L-cysteine instead of BSH.
- The structural and functional characterization of FosB Ef provides a basis for developing novel inhibitors.
- Targeting FosB Ef could enhance the efficacy of fosfomycin in treating resistant enterococcal infections.
