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Title page for ETD etd-07072016-115310

Type of Document Dissertation
Author Keithly, Mary Elizabeth
URN etd-07072016-115310
Title Structure, substrate selectivity, and catalytic mechanism of the fosfomycin resistance enzyme, FosB, from Gram-positive pathogens
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Richard N. Armstrong, Ph.D. (Deceased June 2015) Committee Chair
Walter J. Chazin, Ph.D. Committee Chair
Brian O. Bachmann, Ph.D. Committee Member
Charles R. Sanders, Ph.D. Committee Member
Gary A. Sulikowski, Ph.D. Committee Member
John A. McLean, Ph.D. Committee Member
  • Microbial antibiotic resistance
  • fosfomycin
  • FosB
  • Gram-positive
Date of Defense 2016-04-26
Availability unrestricted
Structure, substrate selectivity, and catalytic mechanism of the fosfomycin resistance enzyme, FosB, from Gram-positive pathogens

By: Mary E. Keithly

Fosfomycin, a broad spectrum antibiotic, is used clinically to treat lower urinary tract infections and gastrointestinal infections and has been suggested as part of a regimen for treatment of multi-drug resistant bacterial infections. However, bacterial fosfomycin resistance enzymes limit the efficacy of the antibiotic. A better understanding of the enzymatic mechanism of fosfomycin resistance can contribute to increasing the efficacy and use of fosfomycin.

One resistance enzyme, FosB, is a Mn2+-dependent thiol-transferase found in Gram-positive bacteria. FosB modifies fosfomycin by catalyzing nucleophilic addition of a thiol, resulting in an inactive compound. In vitro time course kinetic analyses for FosB from four different bacterial strains using L-cysteine and bacillithiol (BSH) reveal a preference for BSH over L-cysteine. Probing metal dependent activation of FosB by Ni2+, Mg2+, Zn2+, and Mn2+ revealed the highest activation of FosB with Mn2+ as the metal cofactor, whereas Zn2+ inhibits FosB enzymes. I concluded that FosB is a Mn2+-dependent BSH-transferase.

Fourteen high-resolution crystal structures of FosB from both Bacillus cereus and Staphylococcus aureus have been determined in complex with various substrates, divalent metals, and products. These structures confirm that FosB is a member of the Vicinal Oxygen Chelate (VOC) superfamily of enzymes. Additionally, a cage of conserved residues orients fosfomycin in the active site such that it is poised for nucleophilic attack by the thiol. The structures also reveal a BSH binding pocket and suggest a highly conserved loop region must change conformation for fosfomycin to enter the active site. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) experiments were utilized to investigate the structural dynamics of FosB. HDX-MS data analysis for this enzyme incubated with various substrates and cofactors indicates that FosB is a highly stable globular protein. Moreover, low signal-to-noise for the conserved loop region made analysis of the dynamics of this area difficult to assess with HDX-MS. These observations suggest nuclear magnetic resonance (NMR) should be applied to investigate the critical loop movement of FosB.

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