The overall objective of this application is to study the structures and functions of enzymes involved in purine and pyrimidine biosynthesis, purine and pyrimidine catabolism, and purine utilization in the biosynthesis of cellular metabolites. Enzymes involved in purine and pyrimidine biosynthesis and metabolism serve as targets for drug design in wide arrange of human diseases including cancer, bacterial infections and parasitic infections. While most of the structures for individual purine and pyrimidine biosynthetic enzymes have been worked out, only a few structures are known for multifunctional enzymes that occur in higher organisms. The multifunctional enzymes may in turn aid in understanding the purinosome, a large multiprotein biosynthetic complex that probably is not amenable to crystallographic analysis. Proposed studies include the vertebrate trifunctional enzyme PurD-PurM-PurN, which catalyzes steps 2, 3 and 5 of purine biosynthesis, and PurD-PurM and PurD-PurM-PurM4-PurN, which are variants found in other higher organisms. We also propose to study OMPDC-OPRT from malaria parasite, which catalyzes steps 6 and 5 of pyrimidine biosynthesis. Catabolic pathways for the degradation of purines and pyrimidines have been previously described and structures are available for the key enzymes;however, recently a new pathway for pyrimidine biosynthesis was discovered in Klebisella pneumoniae and a new pathway was discovered for pyrimidine degradation in Escherichia coli. The discovery of these new pathways was surprising and most of the gene products are both biochemically and structurally uncharacterized. Bioinformatics suggest that a flavoenzyme catalyzes the ring opening in the pyrimidine catabolic pathway - a new catalytic motif in flavoenzymology. The purine catabolic operon encodes two novel enzymatic activities - a putative flavin- dependent uricase and an iron-dependent xanthine oxidase. Finally, analysis of the available genomes indicates many operons associated with uncharacterized cyclohydrolase-catalyzed ring-opening reactions. These reactions are usually associated with purine-derived metabolites such as folate, riboflavin and molypdopterin, suggesting a widespread utilization of purines in the biosynthesis of additional metabolites. We will begin to study purine utilization by examining the biosynthesis of toxoflavin, a good system for understanding the mechanistic enzymology of N-N bond formation. Most of the enzymes required for this research have been cloned and overexpressed. We will determine the structures of these enzymes using X-ray crystallography and in collaboration with Prof. Tadhg Begley study the mechanistic enzymology.
Purine and pyrimidine nucleotides are the building blocks for DNA and RNA. All forms of life depend on these molecules and their levels in the cell are regulated by biosynthesis, import, degradation and metabolism. Understanding these processes offers possible targets for both anticancer and antimicrobial chemotherapies.
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