Mechanistic structure-function studies of the type II citrate synthase of gram-negative bacteria

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Gram-negative bacteria are involved in a wide range of different diseases in plants, animals and humans, with severe economic and health consequences. These range from soft rot in agricultural crops, to contamination of processed foods, and include many of the most dangerous human pathogens (E. coli, N. gonorrhoea, P. aeruginosa, S. typhi, V. cholerae, etc.). Also notable is the fact that Gram-negative bacteria have a unique form of the energy producing enzyme, citrate synthase, designated Type II, whereas most other organisms, including humans, express the Type I dimeric version of this enzyme. Type II citrate synthases form hexamers that are metabolically regulated through strong allosteric inhibition by NADH. As such, these enzymes exhibit a conformational equilibrium between the T (inactive, binding NADH) and R (active, binding substrates) states. In the research program outlined in this application, a strongly synergistic series of mechanistic and inhibitor discovery experiments are proposed. The goal is to not only attain a comprehensive understanding of the unique allosteric function of Type II citrate synthases, but also to explore the possibility of using this knowledge to develop novel anti-bacterials that lock these enzymes into the T inactive state and thereby achieve pathogenic clearance by choking off the energy supply of Gram-negative bacteria. Remote allosteric control as a paradigm for anti-bacterial control is an entirely new approach to the eradication of pathogens, a process that has traditionally depended on direct active site inhibition. We have recently demonstrated the validity of the allosteric lock down approach in a small pilot screening study that identified three new potential anti-bacterials. To build on this success we propose the following mechanistic studies of the Type II citrate synthases: structurally characterizing the R-state; mapping the route of allosteric signal transmission; and, characterizing the nature of allosterically directed active site unfolding. To further the goals of anti-bacterial inhibitor discovery, we further propose to: expand our successful pilot screening program; functionally test identified potential inhibitors; and, structurally characterize the modes of allosteric lock inhibition observed.