Mechanistic Insights into Fosfomycin Resistance: Examination of the FosX Class of Fosfomycin Resistance Proteins

Abstract

The objective of this research was to examine the FosX class of metalloenzymes utilized by pathogenic microorganisms for resistance to the antibiotic fosfomycin. Fosfomycin is an extremely stable natural product produced by strains of soil-dwelling Streptomyces and possesses desirable pharmacological properties. Unfortunately, the existence of fosfomycin-inactivating enzymes endangers the clinical value of this drug. Present research focuses on mechanistic and structural characterization of fosfomycin-inactivating enzymes from the FosA, FosB, and FosX classes of fosfomycin resistance proteins. The well characterized FosA proteins possess robust catalytic activity while the FosB and FosX proteins show significantly less catalytic ability. The FosA and FosB enzymes are thiol-transferases that inactivate fosfomycin through conjugation with glutathione and cysteine, respectively. The more distantly related enzymes of the FosX class, however, catalyze the hydrolysis of fosfomycin. Comparison of FosX proteins from diverse microorganisms reveals a range of catalytic activity, catalytic promiscuity, and ability to confer resistance to fosfomycin in a model organism (E. coli). Critical issues that were addressed in this research included determination of enzyme activity both in vitro and in the biological setting of E. coli, determination of metal binding kinetics, and elucidation of catalytically important resdiues through site-directed mutagenesis studies. Although it is beyond the capacity of this research, elucidation of the molecular basis of resistance will aid the development of inhibitors for these antibiotic-inactivating enzymes.