The overall goal of this project is to establish a detailed understanding of the enzymatic steps involved in the histidine biosynthetic pathway, leading ultimately to a knowledge of the organization of enzyme active sites in vivo. Because of the unique substrates and chemistry of the pathway, these steps are potential targets for selective inhibitors or alternate substrates with direct applications in the development of antimicrobial, antifungal or herbicidal agents. This knowledge maybe coupled with the wealth of genetic information about this system, to create new in vivo metabolic pathways, and for this reason, the research will take on a multifaceted approach. The theme of this proposal is to establish the major mechanistic steps involving the three central enzymes preceding the metabolic branch point linking specific aims. 1. Recombinant DNA methods will be used for the subcloning and over-expression of five target enzymes involved in the de novo biosynthesis of histidine in Escherichia coli. Protocols for the isolation and characterization of three of these enzymes will be developed. 2. The catalytic mechanism of the 5'- phosphoribosylformimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'ProFAR) isomerase will be investigated using several isotopically labeled and fluorinated substrate analogs. A general method will be developed for the synthesis of the key substrate 5'-Pro FAR from D-ribose that will allow for the synthesis of 2H, 3H, 13C labeled analogs from commercially available materials. The synthetic method will be extended to a preparation of four fluorinated ribose derivatives of 5'-ProFAR that will serve as substrate analogs and/or mechanism based inhibitors of the 5'- ProFAR isomerase enzyme. 3. A structure will be determined for the product of the N1-(5'-Phosphoribulosyl)-formimino-5-aminoimidazole-4- carboxamide ribonucleotide glutamine amidotransferase reaction, in order to establish a mechanism for its formation, and the nature of the enzyme activity for the protein encoded by the hisF gene. As a direct comparison to other glutamine amidotransferases, the catalytic properties of the glutamine binding site in this enzyme will be analyzed for a covalent adduct via a gamma-glutamyl ester and the amino acid residue involved in this bond will be identified by affinity labeling and peptide sequence analysis. Efforts toward a three dimensional structure for this enzyme will be initiated exploring a variety of conditions to arrive at crystals of the glutamine amidotransferase that are suitable for x-ray diffraction studies. 4. Plasmid DNA clones for the HIS6 and HIS7 genes of Saccharomyces cerevisiae will be isolated by genetic complementation tests in deficient strains of this organism. These target genes code for the analogous genes in the E. coli histidine biosynthetic pathway which are the subject to this proposal. The DNA sequence of these clones will be analyzed and the deduced amino acid sequence will be used for a comparison with the corresponding proteins in eubacteria. Because of the assumed bifunctional nature of the enzyme encoded by the HIS7 gene, a DNA vector will be developed that allows for expression of this protein in E. coli.