Charles P. Postelle Distinguished Professor
Dihydrofolate reductase (DHFR, EC 22.214.171.124) 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.
Grubbs, J., Rahmanian, S., DeLuca, A., Padmashali, C., Jackson, M., and Howell, E.E.(2011) “Thermodynamics and Solvent Effects on Substrate and Cofactor Binding in E. coli Chromosomal Dihydrofolate Reductase,” Biochemistry 50, 3673-3685.
Philip, V., Harris, J., Adams, R., Nguyen, D., Spiers, J., Baudry, J., Howell, E.E., Hinde, R.J. (2011) “A Survey of Aspartate-Phenylalanine and Glutamate-Phenylalanine Interactions in the Protein Data Bank: Searching for Anion-p Pairs,” Biochemistry 50, 2939–2950.
Kamath, G., Howell, E.E. and Agarwal, P.K. (2010) “The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase,” Biochemistry 49, 9078–9088.
Feng, J., Grubbs, J., Dave, A., Goswami, S., Horner, CG., and Howell, E.E. (2010) “Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields aFunctional, Folded Protein Possessing 45 Substitutions,” Biochemistry 49, 7384–7392.
Yahashiri, A., Nimrod, G., Ben-Tal, N., Howell, E.E. and Kohen, A, (2009) “Effect of electrostatic shielding on H-tunneling in R67 dihydrofolate reductase,” ChemBioChem.10, 2620-2623.
Yahashiri, A., Howell, E.E., Kohen, A. (2008) “Tuning of the H-Transfer Coordinate in Primitive versus Well-Evolved Enzymes,” ChemPhysChem 9, 980-982.
Chopra, S., Dooling, R., Horner, C.G., and Howell, E.E. (2008) “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. 283, 4690-4698.
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.