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.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07072016-115310 |
| Date | 08 July 2016 |
| Creators | Keithly, Mary Elizabeth |
| Contributors | Walter J. Chazin, Ph.D., Richard N. Armstrong, Ph.D. (Deceased June 2015), Gary A. Sulikowski, Ph.D., Brian O. Bachmann, Ph.D., Charles R. Sanders, Ph.D., John A. McLean, Ph.D. |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07072016-115310/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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