Office : WLS, F-327B: (865-974-4507)
Lab: WLS, D307: (865-974-2024)
Ph.D. 1982 Lehigh University, Bethlehem, PA
Folate is vitamin B9. Bacteria synthesize folate de novo while humans obtain folate from their diet. Folate metabolism allows transfer of single carbons; an example is formation of thymidylate which is incorporated into DNA. Thus antifolate drugs can be used to treat various diseases, including bacterial infections and cancer.
After studying enzyme kinetics in the test tube for many years, we are now interested in how folate metabolism enzymes work in crowded conditions. We have recently found that weaker binding of dihydrofolate to three entirely different dihydrofolate reductase scaffolds occurs upon addition of numerous osmolytes (small molecules that take up space in a solution) and crowders. We propose that weak interactions between the dihydrofolate substrate and the osmolyte or crowder are harder to remove than interactions between folate and water. This leads to weaker binding of dihydrofolate to dihydrofolate reductase.
While the folate•(osmolyte) n and folate•(crowder)n interactions are weak, they are plentiful due to the high concentration of molecules in the cell. (An analogy considers how easy it would be to walk in the desert (not crowded situation) vs. crossing Times Square at midnight on New Year’s Eve (crowded condition) vs. crossing Times Square under these same conditions where most everyone is wearing Velcro (crowded plus many weak interactions). These 2 different factors, crowding and weak interactions, are being explored in folate metabolism to examine the hypothesis that osmolarity and crowding are key environmental factors that affect enzyme fitness.
We use enzyme kinetics and biophysical approaches to address this situation with purified enzymes. We additionally use genetics to monitor osmotic stress effects on folate metabolism enzymes. We are beginning metabolomics studies of folate metabolism in collaboration with Shawn Campagna (UT Chemistry Dept).
Bhojane, P., Duff, Jr., M.R., Patel, H.C., Vogt, M., and Howell, E.E. (2014) “Investigation of Osmolyte Effects on FolM: Comparison with Other Dihydrofolate Reductases.” Biochemistry 53, 1330-1341.
Timson, M.J., Duff, M.R., Jr., Dickey, G., Saxton, A., Reyes-De-Corcuera, J.I. and Howell, E.E. (2013) “Further Studies on the Role of Water in R67 Dihydrofolate Reductase” Biochemistry 52, 2118–2127.
Duff, Jr., M.R., Grubbs, J., Serpersu, E.H., and Howell, E.E. (2012) “Weak Interactions between Folate and Osmolytes in Solution,” Biochemistry 51, 2309−2318.
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- Pairs,” Biochemistry 50, 2939–2950.
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 a Functional, Folded Protein Possessing 45 Substitutions,” Biochemistry 49, 7384–7392.