Mechanisms of Flavin-Containing Enzymes of Pyrimidine Metabolism
Our laboratory studies the mechanisms of flavin-containing enzymes involved in pyrimidine metabolism. Because pyrimidines are components of nucleic acids, are modified in the maturation of RNA, and are involved in the synthesis of lipids and glycoproteins, pyrimidine metabolism is critical to life. A number of redox reactions using flavins occur in the myriad of pyrimidine interconversions. We are studying several of these in order to elucidate their reaction mechanisms. By knowing the mechanisms in great detail, we will learn how enzymes accelerate reactions and should be able to design specific inhibitors that may be of therapeutic value. Our studies are guided by the philosophy that enzymes should be studied as reactants, at substrate-level concentrations, rather than as catalysts. This enables us to directly observe events at the active site by a number of spectroscopic and kinetic methods. The flavin prosthetic group (a derivative of vitamin B2) assists us greatly in our studies by acting as a spectral reporter group in the active site as it participates in the events of catalysis.
Dihydroorotate Dehydrogenases - The dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate (DHO) to orotate in the de novo biosynthesis of pyrimidines. They are targets, or are being considered as targets, in the treatment of a large number of diseases, including rheumatoid arthritis, malaria, and cancer. We are currently studying DHODs from Homo sapiens, Escherichia coli, and two forms from Lactococcus lactis. While each of these oxidizes DHO with an FMN prosthetic group, they have differing preferences for their oxidizing substrates. We are investigating the mechanism of FMN reduction in these enzymes in order to determine the transition state structure for flavin reduction and how the protein stabilizes this. We are also studying the mechanisms of flavin oxidation by a variety of substrates and hope to determine the factors that cause a given enzyme to prefer one oxidizing substrate (e.g., fumarate) over another (e.g., ubiquinone).
One of our primary means for studying these reactions is stopped-flow spectrophotometry, which provides intrinsic rate constants. These data allow the detection of intermediates and the measurement of kinetic isotope effects. Our kinetic data are supplemented with a variety of spectroscopic information (Raman, single-molecule fluorescence, absorbance, NMR), by the use of substrate analogs, by quantum calculations, and information from thermodynamic measurements. Our studies have already resulted in the identification of new inhibitors which may be developed further into drug candidates. Many of these projects are collaborations with other groups throughout the world.
Dihydrouridyl-tRNA Synthases - After transcription, tRNA is modified extensively in a variety of ways. One of the most common is the reduction of specific uracil moieties to form dihydrouracil. This reaction is catalyzed by a flavoprotein, dihydrouridine synthase (DUS). We have shown that DUS uses FMN, prefers NADPH over NADH, and transfers the proR-hydride of NADPH to the flavin. Interestingly, rapid reaction of nascent tRNA with the reduced enzyme requires at least one as-yet unidentified modification elsewhere on the tRNA molecule; raw transcripts are very poor substrates. This discover hints that there may be a previously unsuspected preferred order to the myriad of tRNA modification reactions.
Thymidylate Synthases - Another flavoprotein involved in pyrimidine metabolism that we have begun to study is a newly discovered form of thymidylate synthase. Thymidylate synthases methylate 2'-deoxyuridine monophosphate with the methylene group of methylenetetrahydrofolate. In "classic" thymidylate synthases, the methylene group is reduced to the methyl oxidation level by the folate. However, the "new" thymidylate synthase apparently accomplishes this reduction by another mechanism, using an FAD prosthetic group and NAD(P)H as the source of reducing equivalents. Because the "new" thymidylate synthase occurs in a number of pathogens, it is an excellent drug target; because the "new" reaction involves a prosthetic group and an additional substrate, a novel mechanism reaction appears likely.