Abstract

Reaction mechanisms will be determined for the family 1A dihydroorotate dehydrogenase (DHOD) from Lactococcus lactis and Enterococcus faecalis, and the family 2 enzymes from Homo sapiens and Escherichia coli. DHOD, the only redox enzyme in pyrimidine biosynthesis, is an attractive target for drug design in the treatment of many diseases, including malaria, arthritis, and Pneumocystis infections in AIDS patients. The mechanism of reduction of the enzyme-bound FMN by dihydroorotate for the two enzymes will be determined under anaerobic conditions through stopped-flow kinetic analyses, including the use of single- and double deuterium substrate isotope effects, solvent isotope effects, and the pH dependence of the rate constants. Mutant L. lactis A and E. coli enzymes will be studied in order to determine the roles that active site residues have in the reaction. The mechanism of oxidation of reduced L. lactis DHOD A by fumarate will be elucidated in stopped-flow experiments that will determine the pH dependence of the reaction, and in double-mixing experiments, the deuterium isotope effects for the transfer of each hydrogen to fumarate. The mechanism(s) of oxidation by quinones of DHOD A and the E. coli enzyme will be determined in stopped-flow experiments utilizing a range of quinone substrates. The ubiquinone isoprenyl chain-length preference of the E. coli enzyme will be determined. Enzyme-ligand interactions will be probed with Raman spectroscopy.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM061087-01A2
Application #
6434165
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Preusch, Peter C
Project Start
2002-01-01
Project End
2006-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
1
Fiscal Year
2002
Total Cost
$201,100
Indirect Cost

  Institution

Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109

  Publications

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-? 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
Rider, Lance W; Ottosen, Mette B; Gattis, Samuel G et al. (2009) Mechanism of dihydrouridine synthase 2 from yeast and the importance of modifications for efficient tRNA reduction. J Biol Chem 284:10324-33
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
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

Showing the most recent 10 out of 22 publications