The goal of this proposal is to determine how the biosynthesis of the essential coenzyme, pyridoxal phosphate (PALP), is regulated and integrated into cellular metabolism. PALP is the coenzyme for scores of enzymes that participate in all phases of amino acid metabolism and may act as an effector molecule in several metabolic pathways unrelated to amino acid metabolism. Because of these diverse roles, cellular levels of PALP must be carefully regulated in all organisms. Preliminary results in bacteria suggest that regulation of PALP biosynthesis may involve novel mechanisms that sense the availability of certain amino acids. In addition, genetic experiments indicate that the bacterial pdx genes, which encode the PALP biosynthetic enzymes, occur at separate chromosomal locations and that at least one member of this pdx regulon is part of a complex operon. In order to learn more about PALP biosynthesis, recently developed genetic and molecular biological approaches will be applied to a model bacterial system. New classes of pdx mutants will be isolated in E. coli by a variety of selections. These pdx mutations will be mapped, physiologically characterized, and related to the PALP biosynthetic pathway. Transcription and translation fusions, regulatory mutations, and POX kinase mutations will be used along with well-characterized regulatory loci in E. coli to analyze the regulation of PALP biosynthesis at the gene control and pathway levels. Concurrently, the detailed structure and regulation of the pdxA-ksgA locus will be determined using molecular biological techniques and compared to results established previously for the complex pdxB-hisT operon. Besides providing detailed information about PALP biosynthesis, these experiments will address several important questions concerning the regulation of cellular metabolism, including whether there are global controls that link PALP biosynthesis, amino acid metabolism, and phosphate availability, how alternative metabolic pathways are utilized, and how complex regulons are controlled. Results from this basic study of PALP biosynthesis might also help in understanding certain human deficiencies in vitamin B6 metabolism, the interaction between human steroid hormones and PALP-dependent enzymes, and the role of PALP in regulating hypothalamopituitary functions.
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