Development of a system to facilitate the study of R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR), which in nature exists as part of an R-factor, is of interest because it is structurally unrelated to chromosomal DHFRs. It also demonstrates a high resistance to the folate analog trimethoprim. A system to facilitate the study of R67 DHFR using site-directed mutagenesis is described. Native R67 DHFR was isolated and analyzed kinetically. Using structural and kinetic information, possible models for R67 DHFR are discussed. A synthetic R67 DHFR gene was constructed. Synthetic R67 DHFR was analyzed to ensure that it maintains the kinetic parameters of the native enzyme. Finally, it was confirmed that R67 DHFR, truncated at Phe-16 by chymotrypsin, retains full kinetic activity.