Please use this identifier to cite or link to this item:
|Title:||Protein Dynamics and Inhibition of 3-Deoxy-arabino-heptulosonate-7-phosphate Synthase (DAHPS)|
|Advisor:||Berti, Paul J.|
|Abstract:||Antibiotic resistance is the ability of microorganisms to overcome the effects of an antibiotic. While the development of antibiotic resistance is a natural process, the overuse and misuse of antibiotics is accelerating it dramatically. It is estimated that by the year 2050, antibiotic resistance will claim 10 million lives per year unless new therapies are developed. The shikimate pathway is an attractive target for antibiotic development as it is found in archaea, bacteria, fungi and plants, but not in mammals. It begins with the condensation of phosphoenolpyruvate and erythrose 4-phosphate in an aldol-like reaction to produce 3-deoxy-arabino-heptulosonate-7-phosphate (DAHP), catalyzed by DAHP synthase (DAHPS). The pathway leads to the aromatic amino acids, phenylalanine, tryptophan, and tyrosine, and a variety of other aromatic compounds. Our lab has previously produced and characterized potent oxime-based inhibitors of DAHPS and the related enzymes KDO8P synthase and NeuB, an N-acetylneuraminate synthase. The DAHPS·DAHP oxime crystal structure showed half-of-sites inhibitor binding to this homotetrameric enzyme, consistent with the residual enzyme activity observed at high inhibitor concentrations. The crystal structures offered no explanation of the half-of-sites binding, showing minimal changes upon inhibitor binding. However, global hydrogen-deuterium exchange revealed large differences in protein dynamics in the presence and absence of substrates and inhibitors. Our lab is working on understanding the protein dynamics of tyrosine-regulated DAHPS to understand its function and inhibition. Recently, it has been difficult to produce active enzyme, though a number of improvements to the procedure have been developed. In addition, new inhibitory motifs were developed for phenylalanine-regulated DAHPS. This includes N-formylhydrazone-based inhibitor fragments. Pyruvate N-formylhydrazone showed inhibitor activity with Ki = 11 ± 3 μM and Ki = 6 ± 3 μM in the presence of glycerol 3-phosphate, indicating that glycerol 3-phosphate occupying the distal end of the active site helps improve inhibitor binding. This molecule will be further characterized, then the full-size DAHP-based inhibitor will be synthesized and characterized.|
|Appears in Collections:||Open Access Dissertations and Theses|
Files in This Item:
|Protein Dynamics and Inhibition of 3-Deoxy-arabino-heptulosonate-7-phosphate Synthase (DAHPS).pdf||2.09 MB||Adobe PDF||View/Open|
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.