Dihydrofolate reductase (DHFR) is an important enzyme in folate metabolism as tetrahydrofolate 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. The cancer chemotherapeutic agent, methotrexate, and the antibacterial agent, trimethoprim, are potent inhibitors or intracellular DHFR and are used in clinical treatments of cancer and bacterial infections. An increased knowledge of the interactions available to the structure of DHFR is critical to more fully appreciate its mechanism of catalysis and inhibition. Therefore, the main objective of this proposal is the isolation, identification and characterization of functional second site revertant proteins deriving from genes coding for inactive or partially active dihydrofolate reductases (DHFRs). The formation obtained from this study will help to delineate the mechanisms available to DHFR (and protein structure in general) in the suppression of mutational effects. Functional second site E. coli dihydrofolate reductase revertants will be obtained by a multistep process. The first step is deletion of the E. coli DHFR chromosomal gene. This is followed by mutagenesis of the mutant DHFR genes carried in the bacteriophage M13mp8 (supplied by Villafranca et al. (Sci. 222:782(1983)). Revertants, characterized by an increased DHFR activity, will be isolated by displaying either the ability to allow the host cell to grow in minimal media not supplemented by adenine, guanine, thymine, glucine and methionine or by the ability to grow in media containing high concentrations of trimethoprim, an inhibitor of DHFR. Subsequently, the single stranded M13-DHFR DNA will be screened by dot-blot hybridization techniques to eliminate any revertants to a wild type DNA sequence. A second screen of the revertants, to measure protein activity, is in situ activity staining of a nondenaturing electrophoretic gel. The genes coding for functional site revertants will then be sequenced to identify the mutation. The protein will be expressed and kinetically characterized. Km and k-cat values will be obtained at ph 5.0 and 7.0 and compared to the wild type DHFR parameters. Finally, the structure of any especially interesting second site revertant DHFRs will be studied by x-ray crystallography in collaboration with Dr. Joseph Kraut (University of California, San Diego). The determination of the mutant enzyme structure will then allow a precise evaluation of the mutational effects.
| Appleman, J R; Howell, E E; Kraut, J et al. (1990) Role of aspartate 27 of dihydrofolate reductase from Escherichia coli in interconversion of active and inactive enzyme conformers and binding of NADPH. J Biol Chem 265:5579-84 |
| Appleman, J R; Howell, E E; Kraut, J et al. (1988) Role of aspartate 27 in the binding of methotrexate to dihydrofolate reductase from Escherichia coli. J Biol Chem 263:9187-98 |
