DAHP Oxime: A Transition State Mimic Inhibitor Of DAHP Synthase

Abstract

<p>The rise of bacterial infections and increase of antibiotic resistant bacteria has become a major problem in the treatment of bacterial infections. The use and overuse of antibiotics, and the inherent ability of bacteria to adapt to their environment, have lead to the emergence of strains that are resistant to all antibiotics. Ideally, new targets for antibacterial drug therapy would be essential to the virulence of most or all bacteria. That is, antibiotics exploiting these targets would have broad spectrum activity. 3-Deoxy-D-arabinoheptulosonate-7- phosphate (DAHP) synthase could be such a target. This enzyme catalyzes the condensation of erythrose 4-phosphate (E4P) and phoshoenolpyruvate (PEP) to form DAHP. The DAHP synthase-catalyzed reaction is the first committed step in the shikimic acid biosynthetic pathway leading to the aromatic amino acids and other secondary metabolites in all bacteria and some parasites. Inhibition of this enzyme would lead to a depletion of aromatic amino acids within the cell, halting new protein synthesis and killing the cells. Our lab has developed a transition state analogue, DAHP oxime, which is a slow binding, potent inhibitor of DAHP synthase. Kinetic characterization of inhibitor binding revealed DAHP oxime to be a competitive inhibitor with an ultimate Ki* of 81 nM. Crystal structures of DAHP oxime bound to DAHP synthase revealed that the inhibitor bound to two of the four subunits. The two unbound subunits remain catalytically competent, suggesting that DAHP synthase may utilize a half-of-sites mechanism during catalysis. We further investigated changes in DAHP synthase dynamics in response to PEP and DAHP oxime binding via solvent hydrogen/deuterium exchange mass spectrometry. DAHP synthase in the unbound form was loosely structured around the surface exposed regions, whereas the X-ray crystal structures appeared to be more fully structured. Binding of both PEP and DAHP oxime introduced different degrees of dynamic stabilization.</p>