This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our laboratory studies the structures and functions of metabolic enzymes. We are interested in the catalytic mechanisms of individual enzymes, protein:protein interactions between enzymes within a pathway, evolution of protein function, and drug design. From the purine and pyrimidine metabolic pathways, monochromatic data will be taken on crystals of two enzymes from the newly discovered catabolic pathway from Klebsiella pneumoniae. KpHpxT 5-hydroxyisourate hydrolase (HIU) crystals that have been soaked with ligands allopurinol, 8-azaxanthine, allantoin, and 2,6-diaminopurine, and on KpHpxQ, 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase, crystals that have been soaked with the ligand allopurinol, will be tested to better characterize the active sites. Prior crystals of the former diffracted to 2 - 2.5 ? and the latter to 1.6 ?2 ?. Five other enzymes in this catabolic pathway will be targets for later data collection trips. Pyrimidine and purine nucleotides are essential building blocks for the synthesis of nucleic acids and can also take part in energy transfer and storage, protein synthesis and signaling. Because of the importance of these molecules, the enzymes in their metabolic pathways represent potential drug targets for the treatment of many conditions including cancer and several types of parasitic infections. In conjunction with our structural studies of the proteins involved in the biosynthesis of the purine-derived antibiotic toxoflavin, data will be taken on crystals of ToxA, which methylates demethlyated toxflavin, as well as TflA, an oxoflavin lyase, which degrades toxoflavin. Preliminary data from our home source indicates that the crystals diffract to better than 2 ?. These studies are a first step in a long range program designed to understand the biosynthesis of purine-derived metabolites. In addition, toxoflavin biosynthesis is a good system to study N-N bond formation, a process found in a significant number of natural products for which the mechanistic enzymology is still poorly understood. For our studies of enzymes in the azinomycin B biosynthetic pathway, crystals of AziC1, a proposed branched-chain amino acid aminotransferase, from Streptomyces sahachiori will be tested for diffraction. No prior structures for enzymes in this pathway have been determined, so future data trips will involve additional pathway members as diffraction quality crystals are produced. Azinomycin B, a complex natural product isolated from Streptomyces griseofuscus, is a naturally occurring antibiotic that shows antitumor activity at nanomolar concentrations and increases life span in P388 leukemic mice. Because the functional groups found in azinomycin B are both unusual and densely assembled, it is of interest to develop an understanding of the biosynthetic strategy utilized in nature and exploit this to develop additional antitumor agents. The lab has published 18 papers on enzymes in the purine and pyrimidine metabolic pathways in conjunction with our collaborator Dr. Tadhg Begley, now at Texas A&M. This work is supported by NIH grant 5R01GM73220. The azinomyacin research represents a new direction for the laboratory in conjunction with Dr. Coran Watanabe of Texas A&M. We have recently submitted an R01 grant application to NIH in support of this project.
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