Elizabeth Howell Professor, Department of Biochemistry, Cellular & Molecular Biology The University of Tennessee F327 Walters Life Sciences 1414 W. Cumberland Ave Knoxville, TN 37996 865-974-4507 lzh@utk.edu

Keywords:
Structure, function, assembly & folding of dihydrofolate reductase; comparison of chromosomal and R-plasmid forms

Research Area:

Description of Research:

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:

(1) 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.

(2) 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?

(3) 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.

(4) 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 (of 50):

• Howell, EE (2005). Searching sequence space: two different approaches to dihydrofolate reductase catalysis. ChemBioChem. 6(4): 590-600.
  


• Jackson M, Chopra S, Smiley RD, Maynord PO, Rosowsky A, London RE, Levy L, Kalman TI, Howell EE (2005). Calorimetric studies of ligand binding in R67 dihydrofolate reductase. Biochemistry. 44(37): 12420-12433.
  


• Bennett BC, Meilleur F, Myles DA, Howell EE, Dealwis CG (2005). Preliminary neutron diffraction studies of Escherichia coli dihydrofolate reductase bound to the anticancer drug methotrexate. Acta Crystallogr D Biol Crystallogr. 61: 574-579.
  


• Strader, M.B., Chopra, S. Jackson, M., Smiley, R.D., Stinnett, L.G., Wu, J., and Howell, E.E. (2004). Defining the Binding Site of Homotetrameric R67 Dihydrofolate Reductase. Biochemistry. 43: 7403-12.
  


• Hicks SN, Smiley RD, Stinnett LG, Minor KH, Howell EE. (2004). Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations. Journal of Biological Chemistry. 279(45): 46995-47002.
  


• Stinnett LG, Smiley RD, Hicks SN, Howell EE. (2004). Catch 222, the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69.. Journal of Biological Chemistry. 279(45): 47003-47009.
  


• Hicks, S.N., Smiley, R.D., Hamilton, J.B., and Howell, E.E. (2003). Role of Ionic Interactions in Ligand Binding and Catalysis of R67 Dihydrofolate Reductase. Biochemistry. 42: 10569-78.
  


• Pitcher III, W.H., DeRose, E.F., Mueller, G.A., Howell, E.E. and London, R.E. (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, R.D., Hicks, S.N., Stinnett, L.G., Howell, E.E., & Saxton, A.M. (2002). Bisubstrate Kinetics using SAS Computer Software. Anal. Biochem. 301: 153-156.
  


• VerBerkmoes, N.C., Strader, M.B., Smiley, R.D., Howell, E.E., Hurst, G.B., Hettich, R.L. and Stephenson Jr., J.L (2002). Intact Protein Analysis for Site-Directed Mutagenesis Overexpression Products: Plasmid-Encoded R67 Dihydrofolate Reductase. Anal. Biochem. 305: 68-81.
  


• Smiley, R.D., Stinnett, L.G., Saxton, A.M., 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, E.E., Shukla, U., Hicks, S.N., Smiley, R.D., 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..