Elizabeth Howell, Ph.D.

Research Statement

Dihydrofolate reductase (DHFR, EC 1.5.1.3) catalyses the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) using NADPH as a cofactor. DHFR is an important enzyme in folate metabolism as generation of THF is required for the synthesis of thymidylate, purine nucleosides, methionine and other metabolic intermediates. Efficient inhibition of DHFR results in blockage of DNA synthesis and consequent cell death. Therefore inhibition of DHFR activity is the basis for cancer chemotherapy treatments utilizing folate analogs (eg. aminopterin, methotrexate). Also inhibition of DHFR by 2,4 diaminopyrimidines (eg. trimethoprim, pyrimethamine) is the basis for clinical treatment of a number of bacterial infections and malaria. For the latter, different affinities of the pathogen and mammalian enzymes for the drug give rise to the selective toxicities observed.

Our interest in DHFR focuses on the structure and mechanism of a novel type II R-plasmid encoded DHFR (R67 DHFR). Neither the overall structure nor the active site of R67 DHFR is homologous with chromosomal DHFR.

Specific questions we are addressing include:

What residues stabilize the transition state in R67 DHFR and have an effect on kcat? What residues help bind substrate and cofactor and have an effect on Km? Are any special kinetic characteristics associated with the high degree of symmetry seen in R67 DHFR? A 222 fold axis of symmetry occurs at the center of the putative active site pore.

The efficiency (kcat/Km) of R67 DHFR is only l00x less than that of chromosomal DHFR from E. Coli. How does R67 DHFR accomplish reasonably efficient catalysis considering its apparent recent origin and broader reaction specificity?

How do the active sites and mechanisms of R67 DHFR and E. Coli chromosomal DHFR compare? This information will allow us the unparalleled opportunity of comparing two entirely different structures which catalyze the same reaction. Underlying principles for transition state stabilization can then be extracted.

How does R67 DHFR assemble into dimers and then into tetramers? Does folding of a predominately a-sheet structure differ from folding in a or mixed a, b structures?

We are addressing these questions using kinetic and physical studies as well as site directed mutagenesis techniques. X-ray crystal structures (in collaboration with Matthews and Xuong, La Jolla, CA) help evaluate our mutant enzymes.

Selected Publications

Chopra, S., Dooling, R., Horner, C.G., and Howell, E.E. (in press) “A Balancing Act:
Net Uptake of Water During Dihydrofolate Binding and Net Release of Water Upon NADPH Binding in R67 Dihydrofolate Reductase,” J. Biol. Chem.  published online December 17, 2007 as DOI:10.1074/jbc.M709443200

Feng, J., Goswami, S. and Howell, E.E. (2008) “R67, the Other DHFR: Rational Design of an Alternate Active Site Configuration,” Biochemistry 47, 555-565.

Krahn, J.M., Jackson, M.,  DeRose, E.F., Howell, E.E. and London, R.E. (2007), “Structure of a Type II Dihydrofolate reductase catalytic complex,” Biochemistry 46, 14878-14888.

Jackson, M.R., Beahm, R., Duvvuru, S., Narasimhan, C., Wu, J., Wang, H.-N., Hinde, R. J. and Howell, E.E. (2007) “A Preference for Edgewise Anion – Quadrupole Interactions Between Aromatic and Carboxylate Amino Acids,” J. Phys. Chem. B  111, 8242-8249.

Bennett, B., Langan, P., Coates, L., Mustyakimov, M., Schoenborn, B., Howell, E. and Dealwis, C. (2006) “Complementary neutron and ultrahigh resolution X-ray diffraction studies of E. coli Dihydrofolate Reductase complexed with methotrexate,” Proceeding of the National Academy of Science, USA 103, 18493-18498.

Chopra, S., Lynch, R., Kim, S.-H., Jackson, M. and Howell, E.E. (2006) “Effects of Temperature and Viscosity on R67 Dihydrofolate Reductase Catalysis,” Biochemistry 45, 6596-6605.

Jackson, M., Chopra, S., Smiley, R.D., Maynord, P. O’N., Rosowsky, A., London, R.E.,
Levy, L., Kalman, T.I., and Howell, E.E. (2005) “Calorimetric Studies of Ligand Binding
in R67 Dihydrofolate Reductase,” Biochemistry 44, 12420-12433.

Bennett, B.C., Meilleur, F., Myles, D.A.A., Howell, E.E., and Dealwis, C.G. (2005) “Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate.” Acta Crystallographica Section D Biological Crystallography 61, 574-579.

Howell, E. E. (2005) “Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis”, ChemBioChem 6, 590-600.

Strader MB, Chopra S, Jackson M, Smiley RD, Stinnett LG, Wu J and Howell EE (2004) Defining the Binding Site of Homotetrameric R67 Dihydrofolate Reductase. Biochemistry 43: 7403-12.

Hicks SN, Smiley RD, Hamilton JB and Howell EE (2003) Role of Ionic Interactions in Ligand Binding and Catalysis of R67 Dihydrofolate Reductase. Biochemistry 42: 10569-78.

Pitcher III WH, DeRose EF, Mueller GA, Howell EE and London RE (2003) NMR studies of the interaction of a Type II Dihydrofolate Reductase with Pyridine Nucleotides Reveal Unexpected Phosphatase and Reductase Activity. Biochemistry 42: 11150-11160.

Smiley RD, Hicks SN, Stinnett LG, Howell EE and Saxton AM (2002)  Bisubstrate Kinetics using SAS Computer Software. Anal. Biochem. 301: 153-156.

VerBerkmoes NC, Strader MB, Smiley RD, Howell EE, Hurst GB, Hettich RL and Stephenson Jr. JL (2002)  Intact Protein Analysis for Site-Directed Mutagenesis Overexpression Products: Plasmid-Encoded R67 Dihydrofolate Reductase. Anal. Biochem. 305: 68-81.

Smiley RD, Stinnett LG, Saxton AM and Howell E E (2002) Breaking Symmetry: Mutations Engineered into R67 Dihydrofolate Reductase, a D2 Symmetric Homotetramer Possessing a Single Active Site Pore. Biochemistry 41: 15664-15675.

Howell EE, Shukla U, Hicks SN, Smiley RD, Kuhn L and Zavodzsky M (2001) One Site Fits Both: A Model for the Ternary Complex of Folate + NADPH in R67 Dihydrofolate Reductase, a D2 Symmetric Enzyme. J. Computer Aided Molecular Design 15: 1035-1052.

Contact Information

Office:
Room F-327A
Walters Life Sciences
Phone: (865) 974-4507

Lab:
Room D-305
Room D-307
Walters Life Sciences
Phone: (865) 974-2768 (D-305)
Phone: (865) 974-2024 (D-307)

Email:lzh@utk.edu