Abstract

The guiding objective of the proposed research is to increase our understanding of the mechanism of action of dihydrofolate reductase (DHFR) in terms of its molecular structure. DHFR is the target of antifolate drugs such as methotrexate, trimethoprim and pyrimethamine, which are useful as antineoplastics and antibiotics. Detailed understanding of the structure of this molecule and how it functions should aid in efforts to design more sophisticated disease-specific inhibitors.
Specific aims i nclude determining the structure of folate (substrate) containing complexes of the enzyme from E. coli and chicken; refinement at high resolution of the R-67-plasmid DHFR; elucidation of the chicken enzyme structure when it is activated by mercurials; and to crystallize the R-plasmid enzyme in a complex with substrate and cofactor.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA017374-14
Application #
3164674
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1978-05-01
Project End
1991-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
14
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of California San Diego
Department
Type
Schools of Arts and Sciences
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093

  Publications

Sawaya, M R; Kraut, J (1997) Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry 36:586-603
Chen, Y Q; Kraut, J; Callender, R (1997) pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy. Biophys J 72:936-41
Lee, H; Reyes, V M; Kraut, J (1996) Crystal structures of Escherichia coli dihydrofolate reductase complexed with 5-formyltetrahydrofolate (folinic acid) in two space groups: evidence for enolization of pteridine O4. Biochemistry 35:7012-20
Reyes, V M; Sawaya, M R; Brown, K A et al. (1995) Isomorphous crystal structures of Escherichia coli dihydrofolate reductase complexed with folate, 5-deazafolate, and 5,10-dideazatetrahydrofolate: mechanistic implications. Biochemistry 34:2710-23
Sawaya, M R; Pelletier, H; Kumar, A et al. (1994) Crystal structure of rat DNA polymerase beta: evidence for a common polymerase mechanism. Science 264:1930-5
Pelletier, H; Sawaya, M R; Kumar, A et al. (1994) Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science 264:1891-903
Chen, Y Q; Kraut, J; Blakley, R L et al. (1994) Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications. Biochemistry 33:7021-6
Brown, K A; Howell, E E; Kraut, J (1993) Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase. Proc Natl Acad Sci U S A 90:11753-6
McTigue, M A; Davies 2nd, J F; Kaufman, B T et al. (1993) Crystal structures of chicken liver dihydrofolate reductase: binary thioNADP+ and ternary thioNADP+.biopterin complexes. Biochemistry 32:6855-62
McTigue, M A; Davies 2nd, J F; Kaufman, B T et al. (1992) Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin. Biochemistry 31:7264-73

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