News and Publications

Enzyme Structures and Mechanistic Analysis

J.M. Whiteley, K.I. Varughese, C.-F. Chang, T. Bray

The overall program in this laboratory is directed toward structural and mechanistic understanding at the molecular level of selected proteins that function in important eukaryotic (human) metabolic processes or that are critical to the life cycle of bacterial or parasitic species. In the first case, a greater understanding of these proteins may enable us to rectify inherited or acquired (i.e., associated with disease) functional errors and thus increase host survival. In the second case, the ultimate goal is destruction of the potentially infectious species by drugs with enhanced efficacy.

In accordance with these aims, human enzymes categorized as short-chain dehydrogenases/reductases, including dihydropteridine reductase, sepiapterin reductase, 3ß-hydroxysteroid dehydrogenase ^5-4 isomerase, and prostaglandin dehydrogenase are being investigated. In the bacterial and parasitic projects, isolation and structural characterization of the components of the phosphorelay pathway that controls sporulation in Bacillus subtilis have revealed critical features that increase the general understanding of signaling mechanisms in bacteria. In addition, this information indicates a new target for antibiotic design, and we have made a first tentative attempt to design drugs to block this pathway. This approach differs significantly from current therapy and may be of value, because many bacteria are becoming resistant to established drug protocols.

PTR1, a protein derived from Leishmania tarentolae that has pteridine reductase activity, is also being investigated. PTR1 is classified as a short-chain dehydrogenase/reductase, but it differs from dihydropteridine reductase. In L tarentolae, the presence of PTR1 enables the parasite to resist the action of the known chemotherapeutic agent methotrexate. Knowledge of the structure and exact mechanism of action of this enzyme could lead to the design of chemotherapeutic agents that bypass or eliminate this resistance to methotrexate and are toxic to the parasite either alone or in combination therapy. This understanding could have informative value, because another member of the same genus, Leishmania major, causes a widespread human tropical disease and is difficult to eliminate with existing therapy.

Using modern cloning technology, we overexpressed in Escherichia coli each of the proteins that participate in the previously described biological pathways. Each protein has been isolated on a large scale (~100 mg) and purified to homogeneity, and most have been crystallized. Using x-ray crystallography, we fully characterized many of the structures and are analyzing others. With continued investigation, the complete enzymatic functions of the proteins will be known at the molecular level, allowing the design of specific regulatory agents that could improve current therapeutic protocols.

PUBLICATIONS

Barrett, J.F., Goldschmidt, R.M., Lawrence, L.E., Foleno, B., Chen, R., Demers, J.P., Johnson, S., Kanojia, R., Fernandez, J., Bernstein, J., Licata, L., Donet, A., Huang, S., Hlasta, D.J., Macklay, M.J., Ohemeng, K., Frechette, R., Fresca, N.B., Klaubert, D.H., Whiteley, J.M., Wang, L., Hoch, J.A. Antibacterial agents that inhibit two-component signal transduction systems. Proc. Natl. Acad. Sci. U.S.A. 95:5317, 1998.

Grimshaw, C.E., Huang, S., Hanstein, C.G., Strauch, M.A., Burbulys, D., Wang, L., Hoch, J.A., Whiteley, J.M. Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis. Biochemistry 37:1365, 1998.

Madhusudan, Zapf, J., Hoch, J.A., Whiteley, J.M., Xuong, N.X., Varughese, K.I. A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: Crystal structure of Spo0F from Bacillus subtilis. Biochemistry 36:12739, 1997.

Zapf, J., Madhusudan, Grimshaw, C.E., Hoch, J.A., Varughese, K.I., Whiteley, J.M. Origin of response regulator autophosphatase activity: The critical role of a residue adjacent to the active site. Biochemistry, in press.