The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors

Fosfomycin is a broad-spectrum antibiotic that has been underused due to the nature of the resistance mounted against it by various microorganisms. The work presented here elucidates the structure function relationship between the resistance enzyme, FosA, from Pseudomonas aeruginosa, and its substra...

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Main Author:	Brown, Daniel W.
Other Authors:	Professor F. Peter Guengerich
Format:	Others
Language:	en
Published:	VANDERBILT 2010
Subjects:	Chemistry
Online Access:	http://etd.library.vanderbilt.edu/available/etd-05242010-103718/

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spelling	ndltd-VANDERBILT-oai-VANDERBILTETD-etd-05242010-1037182013-01-08T17:16:39Z The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors Brown, Daniel W. Chemistry Fosfomycin is a broad-spectrum antibiotic that has been underused due to the nature of the resistance mounted against it by various microorganisms. The work presented here elucidates the structure function relationship between the resistance enzyme, FosA, from Pseudomonas aeruginosa, and its substrate, glutathione. The relationship between enzyme and substrate is postulated by computational modeling and verified by mutagenesis and kinetic studies. The evolutionary relationship between the P. aeruginosa FosA enzyme and a structurally related but functionally divergent enzyme, the FosX enzyme from Mesorhizobium loti, is then explored using rational mutagenesis and DNA shuffling techniques as well as x-ray crystallography. Finally, the development and use of a high-throughput screen for the discovery of bioactive inhibitors of the P. aeruginosa FosA is described. Using a purified enzyme assay, several new chemical scaffolds have been discovered that effectively inhibit the FosA resistance enzyme. Two of these compounds show activity suggesting bioactivity and specificity. Professor F. Peter Guengerich Professor Alan R. Brash Professor David W. Wright Professor Brian O. Bachmann Professor Richard N. Armstrong VANDERBILT 2010-06-04 text application/pdf http://etd.library.vanderbilt.edu/available/etd-05242010-103718/ http://etd.library.vanderbilt.edu/available/etd-05242010-103718/ en 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.
collection	NDLTD
language	en
format	Others
sources	NDLTD
topic	Chemistry
spellingShingle	Chemistry Brown, Daniel W. The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
description	Fosfomycin is a broad-spectrum antibiotic that has been underused due to the nature of the resistance mounted against it by various microorganisms. The work presented here elucidates the structure function relationship between the resistance enzyme, FosA, from Pseudomonas aeruginosa, and its substrate, glutathione. The relationship between enzyme and substrate is postulated by computational modeling and verified by mutagenesis and kinetic studies. The evolutionary relationship between the P. aeruginosa FosA enzyme and a structurally related but functionally divergent enzyme, the FosX enzyme from Mesorhizobium loti, is then explored using rational mutagenesis and DNA shuffling techniques as well as x-ray crystallography. Finally, the development and use of a high-throughput screen for the discovery of bioactive inhibitors of the P. aeruginosa FosA is described. Using a purified enzyme assay, several new chemical scaffolds have been discovered that effectively inhibit the FosA resistance enzyme. Two of these compounds show activity suggesting bioactivity and specificity.
author2	Professor F. Peter Guengerich
author_facet	Professor F. Peter Guengerich Brown, Daniel W.
author	Brown, Daniel W.
author_sort	Brown, Daniel W.
title	The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
title_short	The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
title_full	The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
title_fullStr	The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
title_full_unstemmed	The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors
title_sort	evolution of fosfomycin resistance enzyme, fosa, from pseudomonas aeruginosa and the development of a high-throughput screen for the discovery of bioactive inhibitors
publisher	VANDERBILT
publishDate	2010
url	http://etd.library.vanderbilt.edu/available/etd-05242010-103718/
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The Evolution of Fosfomycin Resistance Enzyme, FosA, from Pseudomonas aeruginosa and the Development of a High-Throughput Screen for the Discovery of Bioactive Inhibitors

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