MurA and EPSPS are the only known members of the enolpyruvyl transferase family of enzymes. They are attractive targets for the development of new antibiotic drugs. The MurA reaction forms the first committed step in the biosynthesis of the bacterial cell wall. EPSP synthase is the sixth enzyme in the shikimate pathway towards the synthesis of aromatic amino acids and of other aromatic compounds in numerous microorganisms and plants. Both these pathways are absent from mammals but essential for microbial growth. The central hypothesis to the proposed project is that the use of selected mutant enzymes of MurA and EPSPS together with substrate, product and intermediate state analogs will allow for a complete understanding of the entire reaction pathway of enolpyruvyl transfer, starting from free enzyme to the enzyme products complex. The rationale for the proposed research is that once the prerequisites for the enzymatic reaction are identified, this information will be ultimately utilized to design potent inhibitors that selectively target both enzymes. In order to test our hypothesis and accomplish the objective of this project, two specific aims, which integrate techniques such as protein crystallography, enzyme kinetics, fluorescence spectroscopy, molecular cloning, and synthetic organic chemistry, will be pursued: (i) the identification of the prerequisites for the catalysis of enolpyruvyl transfer, and (ii) the identification of the prerequisites for the induced-fit mechanism. These studies will substantially contribute to our understanding of the relationship between the structure and activity of enolpyruvyl transferases. Moreover, they will provide a solid basis for the rational design of novel broad-spectrum antimicrobial drugs targeting these important enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070633-05
Application #
7393225
Study Section
Special Emphasis Panel (ZRG1-BIO (01))
Program Officer
Jones, Warren
Project Start
2005-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2009-03-31
Support Year
5
Fiscal Year
2008
Total Cost
$283,317
Indirect Cost
Name
H. Lee Moffitt Cancer Center & Research Institute
Department
Type
DUNS #
139301956
City
Tampa
State
FL
Country
United States
Zip Code
33612
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Pollegioni, Loredano; Schonbrunn, Ernst; Siehl, Daniel (2011) Molecular basis of glyphosate resistance-different approaches through protein engineering. FEBS J 278:2753-66
Han, Huijong; Yang, Yan; Olesen, Sanne H et al. (2010) The fungal product terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA) . Biochemistry 49:4276-82
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Priestman, Melanie A; Healy, Martha L; Funke, Todd et al. (2005) Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate. FEBS Lett 579:5773-80