Dihydrofolate reductase (DHFR) is an important enzyme in folate metabolism as tetrahydrofolate is require for the synthesis of thymidylate, purine necleosides, 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 of 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 DHRFs. The information obtained from this study will help to delineate the mechanisms available to DHRF (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 mutagenesis of the mutant DHFR genes carried in the bacteriophage M13mp8 (supplied by Villafranca et al., (Science 222:782 (1983)). Revertants, characterized by an increased DHFR activity, will be isolated by their ability to confer growth in media containing high concentrations of trimethoprim, an inhibitor of DHFR. Alternatively a genetic selection using our recently constructed fol minus strain of E. coli (where the DHRF gene has been deleted) may be feasible. Subsequently the single stranded M13-DHFR DNA may 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 sequences to identify the mutation. The protein will be expressed and kinetically characterized. Km and kcat values will be obtained from pH 5-9 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 Drs. E. Villafranca and J. Kraut (Agouron Pharmaceuticals, UCSD). 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 |
