Quorum sensing regulates a host of bacterial behaviors including virulence, biofilm formation, toxin and antibiotic production, luminescence, and motility. Different species of bacteria use a furanosyl derivative as the type 2 autoinducer (AI-2) for interspecies communication. AI-2 is biosynthesized from S-adenosylmethionine via three enzymatic steps. The long-term objectives of this project are (1) to determine the catalytic mechanisms of enzymes involved in bacterial quorum sensing, and (2) to develop specific inhibitors against the synthesis and/or detection of AI-2 as novel antibacterial agents. During this grant period, the project will focus on the catalytic mechanism and inhibition of S-ribosylhomocysteinase (LuxS), which catalyzes the last step of AI-2 biosynthesis. LuxS is a new type of non-heme Fe2+containing enzyme and has a novel catalytic mechanism. In Specific Aim 1, putative catalytic intermediates and substrate analogs will be chemically synthesized and kinetically characterized to demonstrate their involvement in the catalytic pathway. The structures of LuxS in complex with these intermediates and analogs will be determined by X-ray crystallography. Specific Aim 2 is to determine the role of the metal ion in catalysis by electronic absorption and magnetic circular dichroism spectroscopies. Specific Aim 3 is to determine the function of conserved active-site residues in catalysis by site-directed mutagenesis and kinetic and spectroscopic characterization of the mutants. In Specific Aim 4, mechanism-based and metal-chelating inhibitors will be designed and synthesized. The inhibitors will be tested for inhibition of LuxS and antibacterial activity. The mechanism-based inhibitors will also serve as mechanistic probes for studying the LuxS mechanism. Finally, Specific Aim 5 is to purify and identify additional receptor(s) for AI-2.