The long-term goal of this project is to understand the mechanisms that regulate bacterial membrane lipid biosynthesis and explore the structure, function and diversity of the enzymes involved in this pathway. The study of Escherichia coli has historically served as the paradigm for bacterial lipid metabolism. The evolution of lipid biosynthesis as a focal point for the development of novel therapeutics and the availability of a wealth of genomic sequences has stimulated the exploration of these pathways in important pathogens. The discovery of two novel enoyl-[acyl carrier protein] reductases in Gram-positive bacteria during the last grant period highlights the importance of this avenue of research. We have developed the tools for a multidisciplinary attack on this important problem that will incorporate the techniques of structural biology into all facets of the research. The research plan builds on the important discoveries made during the last grant period and is organized into three subject areas. The enoyl reductase step is a key regulator of fatty acid elongation and the target for widely used antibacterial agents. In the first aim, we will investigate the biochemical mechanism, structure and function of the enoyl reductase and expand this work to include the two newly discovered enoyl reductases of Gram-positive bacteria as well as the universally expressed and highly conserved (3-ketoacyl-[acyl carrier protein] reductase. Lipid metabolism is a vital facet of bacterial physiology and in the second aim we will define the regulatory mechanisms that integrate fatty acid biosynthesis into cell physiology and coordinate membrane lipid formation with macromolecular biosynthesis. Our investigation of fatty acid biosynthesis in Gram-positive bacteria will focus on elucidating the pathways for unsaturated fatty acid synthesis in an important pathogen. The condensing enzymes are key regulators of fatty acid composition, and in the third aim, we will define the molecular characteristics that determine their substrate specificity, use these enzymes as a model for defining the critical 3- dimensional features necessary for the docking of acyl carrier protein, and determine the mechanism of action of a broad-spectrum antibiotic, thiolactomycin. The results of these investigations will provide important new information on the structure, function, diversity and regulation of fatty acid biosynthesis that will contribute to the basic understanding of bacterial physiology and complement the development of novel antibacterialtherapeutics.
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