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. 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. We have undertaken structural studies of various enzymes that play roles in the metabolism of pyrimidines and purines. Adenosine kinase (AK), a key enzyme in purine metabolism in parasites and a potential chemotherapeutic target for the treatment of Toxoplasma gondii infections, catalyzes the ATP dependent phosphorylation of adenosine. Purine nucleoside phosphorylase (PNP), which catalyzes the reversible phosphorolysis of ribonucleosides and 2'- deoxyribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate, is an important enzyme for the salvage of purine nucleotides. RutA is a FMN dependent mono-oxygenase involved in a recently discovered pyrimidine degradation pathway that converts uracil (or thymine) to 3-hydroxypropionate (or 2-methyl-3-hydroxypropionate) in E. coli. Uridine phosphorylase (UP) catalyzes the reversible phosphorolysis of uridine with the formation of ribose-1-phosphate and uracil. Orotidine-5'-phosphate decarboxylase orotate phosphoribosyltransferase (OMPDC-OPRT) is a bifunctional enzyme that catalyzes the last two steps in the synthesis of uridine-5'-monophosphate (UMP). In addition to their many cellular uses, some organisms can metabolize nucleotides as a nitrogen source. Recent studies by two groups on Klebsiella sp. have revealed a gene cluster that is responsible for expressing the enzymes for utilizing purines as a sole nitrogen source in this organism. We have structurally characterized several of the enzymes that catalyze the breakdown of hypoxanthine to allantoin in Klebsiella pneumoniae in order to better understand this interesting pathway. Pseudouridine is the C-glycoside isomer of uridine and is the most abundant modification in RNA. It ubiquitously exists in tRNA, rRNA, snRNA and snoRNA. Recently, various biochemical, biophysical and genetic studies characterized two enzymes that are predominantly responsible for the biosynthesis of pseudouridine. Pseudouridine kinase (YeiC) phosphorylates pseudouridine to pseudouridine-5'-phosphate and pseudouridine glycosidase (YeiN) catalyzes the conversion from pseudouridine-5'-phosphate to uridine and ribose-5-phosphate by cleaving the C-C glycosidic bond. Structural studies of these enzymes will provide insights into the mechanism of pseudouridine biosynthesis and provide tools for the screening of possible antibacterial drugs that target this pathway. Mildiomycin is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease on plants and is used commercially as a fungicide. The initial steps of mildiomycin biosynthesis involve the cleavage of 5-hydroxymethyl cytidine-5'-monophosphate at the glycosidic bond by the enzyme MilB. It has been shown that MilB also catalyzes the cleavage reaction for cytidine-5'-monophosphate. The crystal structure of MilB was solved to better understand how its structure varies from other nucleotide hydrolases and how it provides its substrate specificity. Toxoflavin is an azapteridine that is poisonous to many plants, fungi, animals, and bacteria. Recently, toxoflavin has gained increasing interest because infection of rice plants by toxoflavin-producing bacteria such as Burkholderia glumae has led to a substantial loss of rice crops in the United States and Asia. Several of the steps in the biosynthesis of toxoflavin involve uncharacterized proteins that may potentially exhibit novel chemistry. Specifically, the proteins ToxC and/or ToxD appear to catalyze nitrogen-nitrogen bond formation which is poorly understood and the protein ToxA is predicted to catalyze sequential transmethylation reactions in the final step of toxoflavin production. To combat the toxicity of toxoflavin, recent work has exploited the ability of some enzymes to utilize dioxygen in the biosynthesis and biodegradation of numerous aliphatic and aromatic compounds. Recent work has led to the successful production of transgenic rice plants which express the putative oxygenase toxoflavin lyase (TflA) of Paenibacillus polymyxa JH2 to combat the deleterious effects of toxoflavin-producing bacteria on rice.
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