The research proposed will provide a detailed characterization of a newly discovered class of proteins, the long-chain N-acyl amino acid synthases. Four aspects of these enzymes will be explored. 1- Enzymatic selectivity for amino acid and fatty acid substrates will be studied by assays on reconstituted enzyme produced by over-expression in E. coli. 2-A high-resolution model of the enzyme will be determined through X- ray crystallography on a selenomethionine-containing derivative of an N-aceyl amino acid synthase. 3-Structural determinants of substrates will be rationally modified by the use of site-directed mutagenesis based on careful analysis of the X-ray model. 4- The genetic diversity and distribution of these enzymes among soil microorganisms will be assessed through techniques involving the hybridization of environmental DNA (eDNA) libraries with a DNA probe designed to detect sequences required for enzymatic activity in the N-acyl amino acid synthases. This research will not only provide a greater understanding of structural aspects of substrate recognition and catalysis for this new class of proteins, but will also provide a means of detecting new biosynthesis pathways incorporating similar enzymatic activity through the DNA probes produced. The ability to design genetic probes of protein families with few known sequences greatly extends the utility of heterologous expression as a means of studying biosynthetic pathways in unculturable microbes.
|Van Wagoner, Ryan M; Clardy, Jon (2006) FeeM, an N-acyl amino acid synthase from an uncultured soil microbe: structure, mechanism, and acyl carrier protein binding. Structure 14:1425-35|