Allosteric citrate synthase

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The enzyme citrate synthase plays a central role in the production of metabolic energy in almost all organisms. In most organisms, including humans, the enzyme catalyzes its reaction as quickly as possible, with no braking mechanism as long as the raw materials are available. In one large class of bacteria, the Gram-negative bacteria, the situation is more complex: under certain conditions, it can be inhibited (shut down completely) by a biological reducing agent, NADH. To make this regulation possible, Gram-negative citrate synthase has evolved to a larger enzyme which can exist in either an active form, or in a second form which interacts with NADH and is inactive. We have been able to use the method of X-ray crystallography to determine the complete structure of Gram-negative citrate synthase in the NADH-binding or inactive form. In the present research, we wish to use molecular biology methods to force this enzyme into the active form, and determine that structure. Our second aim is to exploit the fact that Gra-negative citrate synthase can be turned off, while the human enzyme cannot, to design new antimicrobial drugs. Some of the most dangerous bacterial pathogens (like E. coli, involved in some kinds of food and water poisoning, and Pseudomonas aeruginosa, involved in lung infections in cystic fibrosis) are Gram-negatives. We are using high-throughput screening of chemical libraries to identify novel compounds that can inhibit Gram-negative citrate synthase, as NADH does, and then refine these discoveries in the hope of producing antimicrobials that can turn off Gram-negative bacterial growth in the case of infections, while having no effect on the human enzyme.