Mentor: Bruce Palfey
Thesis Defense: November 5, 2008
Flavoenzymes involved in pyrimidine oxidation and reduction
Pyrimidines are chemical compounds found throughout nature. They occur in DNA, RNA, antibiotics, and a myriad of other natural products. One of the many chemical alterations found to occur during the biosynthesis, degradation, and modification of pyrimidines is that of carbon-carbon bond reduction or oxidation.
The majority of the enzymes known to perform this reaction are flavo-enzymes, which catalyze either the reduction or oxidation of the carbon-carbon bond at the C5 and C6 positions of the pyrimidine ring. Enzymes from two of these classes are studied here, the dihydroorotate dehydrogenases and the dihydrouridine synthases.
Dihydroorotate dehydrogenases catalyze the formation of a carbon double-bond in the biosynthesis of uracil, by oxidizing dihydroorotate to orotate. The kinetic mechanism for the dihydroorotate dehydrogenase from Enterococcus faecalis was investigated. The kinetic rate constants for the oxidation of dihydroorotate, the off rate for the product orotate, and the oxidative rate constant for the oxidation by fumarate were determined at pH 8.5, 4 °C. The steady state kinetics of the reaction were also determined. These data indicate that the oxidation of the enzyme by fumarate is the rate limiting step for the enzyme. The binding constants for the ligands fumarate, succinate, orotate, and dihydroorotate were determined as well as the binding constants for the ligands 3,4-dihydroxybenzoate and 3,5-dihydroxybenzoate.
The effect of pH upon the reaction was investigated. The results of these experiments indicate a variable pKa affecting the oxidative and reductive half-reactions. This pKa seems to be affected by the presence of different substrates in the active site during each half-reaction. The pKa changes from 8.3 for the reductive half-reaction to 7.5 for the oxidative half-reaction. The origin of the pKa is most likely an active site cysteine. A second pKa above 12 was also observed, affecting the binding of the ligands fumarate, orotate, and dihydroorotate to the enzyme. This pKa is attributed to the active site lysine, lysine 55.
Crystal structures of this enzyme with dihydroorotate, L-malate, 3,4-dihydroxybenzoate, 3,5-dihydroxybenzoate, and orotate were obtained. Little variation is seen between one ligand-bound form and another. The major changes seen between the different ligand-bound forms of the enzyme occur in the active site loop. Some ligand-bound enzyme complexes show a greater or lesser mobility of this loop.
Three dimensional NMR experiments show that the enzyme is more dynamic in solution than the crystal structures indicate. Upon binding of ligands large resonance shifts are seen in the NMR spectra, indicating changes in the protein structure. These experiments also elucidated a static core of residues whose resonances are unchanged upon binding to ligands.
Dihydrouridine synthases (DUSs) perform an analogous but opposite reaction to dihydroorotate dehydrogenases. They reduce the double bonds in uridine rings forming dihydrouridine at specific sites in tRNA. The dihydrouridine synthase from Thermotoga maritima and the DUS 2 enzyme from Sacharomyces cerevisiae were studied. Both enzymes were shown to oxidize NADPH in a proR specific fashion. The kinetic rate constants for the reduction of both enzymes by NADPH, and their oxidation by tRNA, were determined. The T. maritima enzyme showed very slow reductive and oxidative rate constants. By analyzing kinetic isotope effects for the reductive half-reaction it was concluded that this slow reactivity is due to a slow isomerization of the enzyme. This is most likely due to the experiments having been performed 30 degrees below the enzymes expected temperature optimum, an unfortunate necessity given the enzymes instability at higher temperatures.
Examination of the kinetic rate constants for the oxidation of the DUS 2 enzyme from S. cerevisiae shows that this enzyme functions more rapidly upon modified tRNA than on in vitro -transcribed tRNA. This indicates that this enzyme is dependent upon prior modification of the substrate tRNA for proper activity. Through mutagenesis studies we also determined that an active site cysteine, cysteine 117, plays a role in the reduction of the tRNA by the enzyme, likely acting as an active site acid.
Current Position: Patent Examiner, US Patent & Trademark Office, Alexandria, VA
Undergraduate Institution: Purdue University
My research centers on flavoenzymes which oxidize and reduce pyrimidine rings. I have studied two types of enzymes called dihydroorotate dehydrogenases and dihydrouridine synthases. Dihydroorotate dehydrogenases oxidize dihydroorotate to form orotate in the de novo pyrimidine synthetic pathway. The protein which I study is from the pathogen Enterococcus faecalis, one of the major causes of nosocomial disease. The majority of this project is centered on understanding the dynamics of the enzyme through kinetics, crystallography, and 3D NMR.
Dihydrouridine synthases reduce the pyrimidine rings of specific uracils in tRNA. These enzymes are a relatively new family of enzymes and the majority of this project has been determining the requirements necessary for enzymatic function. I have examined the kinetics of the enzymes from Thermotoga maritima and Saccharomyces cerevisiae. The experiments have determined a need for prior modification of the substrate tRNA for efficient catalysis.
Rider LW, Ottosen MB, Gattis SG, Palfey BA. Mechanism of dihydrouridine synthase 2 from yeast: the Importance of modifications for efficient tRNA reduction. J. Biol. Chem.., 284, 10324-10333
Meetings and Presentations
Chemistry Biology Interface Symposium, April 2007, "Understanding Dihydrouridine Synthases"
Rustbelt RNA Meeting, September 2006, "Understanding Dihydrouridine Synthase from T. maritima"
Midwest Enzyme Chemistry Conference, September 2006, "Understanding Dihydrouridine Synthase from T. maritima"
American Chemical Society Great Lakes Regional Meeting, May 2006, "Characterization of T. maritima Dihydrouridine Synthase"
Chemistry Biology Interface Symposium, May 2006, "Characterization of T. maritima Dihydrouridine Synthase"
Midwest Enzyme Chemistry Conference, September 2005, "Characterization and Inhibition of Class 1A Dihydroorotate Dehydrogenase from Enterococcus faecalis"
Chemistry Biology Interface Symposium, May 2005, "Kinetics and Inhibition in Enterococcus faecalis Dihydroorotate Dehydrogenase"