One Site Fits Both: Catalysis in R67 Dihydrofolate Reductase

University Of Tennessee Knoxville, Knoxville TN

Investigators

Abstract

Dihydrofolate reductase (DHFR) is an important enzyme in folate metabolism as its reaction product, tetrahydrofolate, is required for the synthesis of numerous metabolic intermediates. Inhibition of DHFR results in blockage of DNA synthesis, leading to cell death. Type II DHFR, typified by R67 DHFR, is unrelated genetically and structurally to chromosomal DHFRs. The active R67 DHFR is a homotetramer, with a toroidal (doughnut) shape and a single pore that contains a single active site. The presence of only one active site per oligomer is because only two molecules can bind concurrently to the active enzyme, most likely due to steric constraints. Among several possible complexes, the productive complex is the one containing the substrate (dihydrofolate) and cofactor (NADPH) bound to the active enzyme. The constraints imposed by the symmetry of the oligomer as well as the absence of catalytic bases suggest R67 DHFR may be a primitive enzyme. R67 DHFR's ability to form multiple ligand complexes as well as the dual roles for several active site residues supports the hypothesis that R67 DHFR uses a variation of 'hot spot' binding which is focused in an enzyme active site rather than at protein-protein interfaces. The hot spot strategy is an excellent design for a primitive enzyme to adopt. A drawback of the symmetry is that binding to NADPH and dihydrofolate cannot be independently optimized. To further evolve R67 DHFR function would require a gene duplication event, followed by divergence of the gene copies. In this project, recombinant DNA technology has been used to produce quadruplicated R67 DHFR gene, resulting in a single polypeptide possessing the essential structure of tetrameric R67 DHFR. The goal of this research is to introduce asymmetric mutations that will allow independent manipulation of each half of the active site pore, leading to favorable interactions with NADPH on one side and complementary interactions with dihydrofalate on the other. This strategy may result in a more efficient enzyme.