Pyrimidine metabolism is vital, making it important to understand at the chemical level and making it an excellent target for drug development. We will investigate the reaction mechanisms of dihydroorotate dehydrogenases (DHODs), flavin-dependent enzymes in the biosynthetic pathway, and the evolutionarily related dihydrouridine synthases (DUSs), which reduce specific uracils during the maturation of tRNA. Our goal is to elucidate reaction mechanisms and origins of substrate or ligand specificity in ways ranging from characterizing transition states to uncovering dynamic behavior in catalysis. The results of these studies will facilitate the design of enzyme inhibitors, which may be developed into useful drug candidates. Transition state structures are at the heart of enzymatic catalysis. Previously we found significant differences between the transition states for flavin reduction in Class 1A and Class 2 DHODs, indicating the need for a higher level of understanding. The transition states for flavin reduction in the three classes of DHODs will be probed by measuring 13C and 15N kinetic isotope effects. Stopped-flow experiments will be used to determine deuterium isotope effects on the reduction of a Class 1B DHOD. Complementary stopped-flow and single-molecule studies on a Class 1B DHOD will enable us to dissect factors controlling the chemistry at the flavins, intramolecular electron transfer, and dynamics. We have already discovered two inhibitors that bind specifically to Class 1A DHODs which occurs in some pathogenic bacteria and protozoa. Our kinetic and structural studies suggest a new molecule to be synthesized and studied. However, the reason that our inhibitors do not bind to Class 2 enzymes remains an enigma. Random mutagenesis will be used to create functional Class 1A mutants that are no longer inhibited, and conversely, Class 2 mutants that are inhibited. Interesting enzymes will be studied in detail thermodynamically, kinetically, and structurally. Related chemistry is performed by the DUSs, flavoproteins which are structurally related to DHODs and reduce specific uracil moieties in maturing tRNA. The function of dihydrouracil remains uncertain, but its widespread occurrence suggests an important role, and it has recently been shown to be important in lung cancer. We will determine the substrate specificities of selected model DUSs and probe the interactions of the protein and tRNA by chemical and biophysical means. Vitamin B2 transfers electrons when certain proteins speed chemical reactions that create or modify the building-blocks of DNA or RNA the molecules which carry genetic information. Compounds that specifically interfere with these vital reactions in infectious bacteria could be used as drugs. In order to design such compounds, we will study the reactions of several proteins at a very high level of detail by observing the color changes associated with the vitamin. Our studies of the rates of the chemical reactions will identify important parts of the proteins, how they move during reactions, how they speed the synthesis of products, and how these reactions might be blocked.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061087-09
Application #
7994229
Study Section
Special Emphasis Panel (ZRG1-MSFE-S (01))
Program Officer
Anderson, Vernon
Project Start
2002-01-01
Project End
2011-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
9
Fiscal Year
2011
Total Cost
$279,796
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Prongjit, Methinee; Sucharitakul, Jeerus; Palfey, Bruce A et al. (2013) Oxidation mode of pyranose 2-oxidase is controlled by pH. Biochemistry 52:1437-45
Mishanina, Tatiana V; Koehn, Eric M; Conrad, John A et al. (2012) Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase. J Am Chem Soc 134:4442-8
McDonald, Claudia A; Fagan, Rebecca L; Collard, Francois et al. (2011) Oxygen reactivity in flavoenzymes: context matters. J Am Chem Soc 133:16809-11
McDonald, Claudia A; Palfey, Bruce A (2011) Substrate binding and reactivity are not linked: grafting a proton-transfer network into a Class 1A dihydroorotate dehydrogenase. Biochemistry 50:2714-6
Collard, François; Fagan, Rebecca L; Zhang, Jianye et al. (2011) The cation-π interaction between Lys53 and the flavin of fructosamine oxidase (FAOX-II) is critical for activity. Biochemistry 50:7977-86
Purcell, Erin B; McDonald, Claudia A; Palfey, Bruce A et al. (2010) An analysis of the solution structure and signaling mechanism of LovK, a sensor histidine kinase integrating light and redox signals. Biochemistry 49:6761-70
Sollner, Sonja; Deller, Sigrid; Macheroux, Peter et al. (2009) Mechanism of flavin reduction and oxidation in the redox-sensing quinone reductase Lot6p from Saccharomyces cerevisiae. Biochemistry 48:8636-43
Kow, Rebecca L; Whicher, Jonathan R; McDonald, Claudia A et al. (2009) Disruption of the proton relay network in the class 2 dihydroorotate dehydrogenase from Escherichia coli. Biochemistry 48:9801-9
Koehn, Eric M; Fleischmann, Todd; Conrad, John A et al. (2009) An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature 458:919-23
Fagan, Rebecca L; Palfey, Bruce A (2009) Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli. Biochemistry 48:7169-78

Showing the most recent 10 out of 22 publications