Health care providers are challenged daily by an increasing resistance of pathogenic bacteria to their antibacterial arsenal. To overcome this problem, it is necessary to design new and innovative antibiotics with totally different modes of action so that, no cross-resistance with present agents should occur. Most antimicrobial drugs act by inhibiting key enzymes in the biosynthesis of macromolecular molecules necessary for viability of the microorganism. Success in this type of approach necessitates a thorough understanding of the enzyme(s) at the molecular level. The goal of this work is to collect mechanistic information on the enzymes 3-deoxy-D-mannoo-octulosonate 8-phosphate and 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The information will prove useful in the design of selective inhibitors of these unique enzymes, namely a new generation of mechanistically diverse antibiotics. The goals of this project are to establish 1. The mechanism for the formation of 3-deoxy-D-manno-octulosonic 8-phosphate (KDO 8-P) from arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) catalyzed by the enzyme KDO 8-P synthase (EC 4.1.2. 16), an enzyme involved in the biosynthesis of the lipid A portion of the lipopolysaccharide region of the cell envelope of gram-negative bacteria, 2. The mechanism for the formation 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) from erythrose 4-phosphate (E 4-P) and PEP catalyzed by the enzyme DAH 7-P synthase [EC 4.1.2.15], the enzyme that catalyzes the first committed step in the biosynthesis the aromatic amino acids and various aromatic secondary metabolites. The specific alms focus on the use of diverse techniques to """"""""visualize"""""""" the potential tetrahedral intermediate. These methods include a rapid mixing, pulsed-flow ESIMS technique to confirm the formation of a reaction intermediate(s) and rotational-echo double-resonance NMR experiments of sub-zero substrate entrapped in enzyme to observe the intermediate. A rapid temperature quench methodology will be developed to isolate the potential intermediate(s) for NMR structural studies. Multinuclear NMR analysis of the interaction of the synthases with various labeled substrate analogues will be utilized to observe abortive intermediates and substrate analogs designed to """"""""stabilize"""""""" this potential abortive intermediate(s) will be used to further understand the mechanisms of these reactions. The role of the metal ion will also be investigated. Site-directed mutagenesis studies, based on x-ray crystallographic data, will be exploited to gain further insight into the contribution of enzyme functionalities to substrate binding, monomer interface interactions and to the mechanism of the enzyme.
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