dc.creator | Keithly, Mary Elizabeth | |
dc.date.accessioned | 2020-08-22T17:18:25Z | |
dc.date.available | 2016-07-08 | |
dc.date.issued | 2016-07-08 | |
dc.identifier.uri | https://etd.library.vanderbilt.edu/etd-07072016-115310 | |
dc.identifier.uri | http://hdl.handle.net/1803/12847 | |
dc.description.abstract | 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. | |
dc.format.mimetype | application/pdf | |
dc.subject | Microbial antibiotic resistance | |
dc.subject | fosfomycin | |
dc.subject | FosB | |
dc.subject | Gram-positive | |
dc.title | Structure, substrate selectivity, and catalytic mechanism of the fosfomycin resistance enzyme, FosB, from Gram-positive pathogens | |
dc.type | dissertation | |
dc.contributor.committeeMember | Gary A. Sulikowski, Ph.D. | |
dc.contributor.committeeMember | Brian O. Bachmann, Ph.D. | |
dc.contributor.committeeMember | Charles R. Sanders, Ph.D. | |
dc.contributor.committeeMember | John A. McLean, Ph.D. | |
dc.type.material | text | |
thesis.degree.name | PHD | |
thesis.degree.level | dissertation | |
thesis.degree.discipline | Chemistry | |
thesis.degree.grantor | Vanderbilt University | |
local.embargo.terms | 2016-07-08 | |
local.embargo.lift | 2016-07-08 | |
dc.contributor.committeeChair | Walter J. Chazin, Ph.D. | |
dc.contributor.committeeChair | Richard N. Armstrong, Ph.D. (Deceased June 2015) | |