W0451

Quorum Sensing Signal Generation by the AHL Synthsase LasI in Pseudomonas aeruginosa Pathogenesis. T.A. Gould^*, R.C. Murphy^*, H.P. Schweizer^#, M.E.A. Churchill^*, ^*Dept. of Pharmacology, Univ. of Colorado Health Sciences Center, Denver, CO, ^#Dept. of Microbiology, Colorado State Univ., Fort Collins, CO.

Quorum sensing, the cell density dependent regulation of gene expression, allows bacteria to interact with and respond to changes in the environment, and is vital for their survival. Quorum sensing systems in gram-negative bacteria consist of three components: the acyl-homoserine lactone (AHL) synthase, a signal molecule, and a response regulator. The AHL synthase catalyzes the formation of the signal molecule, AHL, from two substrates, S-adenosylmethionine (SAM) and acyl-acyl carrier protein (ACP). The response regulator initiates gene expression once a sufficient concentration of the AHL is present in the local environment.

The human opportunistic pathogen Pseudomonas aeruginosa utilizes quorum sensing to regulate both the virulence and persistence of infection. Inhibition of the Las quorum sensing system in P. aeruginosa prevents mature biofilm formation and attenuates virulence, which results in a marked decrease in the lethality of infection. For these reasons, LasI, the AHL synthase of the Las system, is an attractive target for the design of antibacterial therapeutics.

Understanding the specific interactions of residues involved in binding and catalysis of LasI will greatly improve the likelihood of developing effective inhibitors. LasI crystals were produced using a novel crystal engineering approach and diffract to 2.3 Å. The structure of LasI was solved using Single Isomorphous Replacement with Anomalous Scattering (SIRAS) and refinement of the structure is complete. A detailed analysis of this structure has revealed putative binding sites for acyl-ACP and SAM. Current studies using mutagenesis and mass spectroscopy are aimed at confirming these hypotheses. This information will be integral in furthering efforts to produce specific inhibitors for LasI and other enzymes in this class by structure-based drug design.