Pyrimidine metabolic enzymes have long been recognized as targets in which drug intervention can be effective in slowing the growth of infectious microorganisms or rapidly proliferating cells. In two pyrimidine metabolic pathways -the de novo pathway and the thymidine salvage pathway, a rare pathway apparently present only in some fungi - the final pyrimidine base modification step to yield the uracil ring is a decarboxylation. The two decarboxylases that catalyze these reactions are OMP decarboxylase (ODCase) and iso-orotate decarboxylase (IDCase), respectively. The long-term objective of this project is to contribute to a complete elucidation of the mechanisms of these two decarboxylases, allowing a rational approach to the feasibility of these enzymes as inhibitor sites in pyrimidine metabolism.
The specific aims for the study of ODCase will be to verify whether or not the active site lysine residue (Lys93 of the yeast enzyme), identified recently in crystal structures of four ODCases from different organisms, contacts O2 of the substrate, as originally postulated. This will be addressed using spectroscopic studies of ODCase in complex with various inhibitors: 1) 1H NMR spectroscopy of engineered ODCase with 15N specifically labeled at the active site amino group; 2) NMR spectroscopy of ODCase complexed with inhibitors with 15N, 13C1' and 1H1' and 3) Raman difference spectroscopy of ODCase/inhibitor complexes, to determine whether or not O2 of the pyrimidine nucleotide inhibitor is polarized by hydrogen bonding to the enzyme. The first specific aim for the study of IDCase will be to develop an effective affinity chromatography purification method based on the observed tight binding of the enzyme to 5-nitrouracil. Upon adequate enzyme purification, kinetic measurements will be determined for substrate and inhibitor binding, which may include inhibition by pharmacologically significant 5-fluorinated pyrimidines. Adequate enzyme purification will also allow examination of the mode of binding of 5-nitrouracil using NMR spectroscopy of the IDCase/[13C5] 5-nitrouracil complex. Finally, adequate protein purification will allow N-terminal amino acid sequencing, leading to the design of oligonucleotide probes that will be used in an attempt to locate the IDCase gene in a cDNA library of genes from Rhodotorula glutinis.
| Wepukhulu, Wickliffe O; Smiley, Vanessa L; Vemulapalli, Bhargavi et al. (2008) A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: mechanistic implications. Org Biomol Chem 6:4533-41 |
| Smiley, Jeffrey A; Kundracik, Melisa; Landfried, Daniel A et al. (2005) Genes of the thymidine salvage pathway: thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa. Biochim Biophys Acta 1723:256-64 |
| Krungkrai, Sudaratana R; DelFraino, Brian J; Smiley, Jeffrey A et al. (2005) A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization. Biochemistry 44:1643-52 |
| Smiley, Jeffrey A; DelFraino, Brian J; Simpson, Beth A (2003) Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase. Arch Biochem Biophys 412:267-71 |
