The crystal structures and enzymic properties of two mutant dihydrofolate reductases (Escherichia coli) were studied in order to clarify the functional role of an invariant carboxylic acid (aspartic acid at position 27) at the substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp27 with asparagine; the other is a primary-site revertant to Ser27. The only structural perturbations involve two internally bound water molecules. Both mutants have low but readily measurable activity, which increases rapidly with decreasing pH. The mutant enzymes were also characterized with respect to relative folate: dihydrofolate activities and kinetic deuterium isotope effects. It is concluded that Asp27 participates in protonation of the substrate but not in electrostatic stabilization of a positively charged, protonated transition state.

Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 M(r) homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 A resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 A in length, passing through the middle of the molecule. Two folate molecules bind asymmetrically within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure to chromosomal DHFRs. A recent crystal structure of the active tetrameric species describes a single active site pore that traverses the length of the protein (Narayana et al., 1995). Related sites (due to a 222 symmetry element at the center of the active site pore) are used for binding of ligands, i.e., each half-pore can accommodate either the substrate, dihydrofolate, or the cofactor, NADPH, although dihydrofolate and NADPH are bound differently. Ligand binding in R67 DHFR was evaluated using time-resolved fluorescence anisotropy and isothermal titration calorimetry techniques. Under binary complex conditions, two molecules of either NADPH, folate, dihydrofolate, or N10 propargyl-5,8-dideazafolate (CB3717) can be bound. Binding of NADPH displays negative cooperativity, binding of either folate or dihydrofolate shows positive cooperativity, and binding of CB3717 shows two identical sites. Any asymmetry introduced by binding of one ligand is proposed to induce the cooperativity associated with binding of the second ligand. Evaluation of ternary complex formation demonstrates that one molecule of folate binds to a 1:1 mixture of R67 DHFR+NADPH. These binding results indicate a maximum of two ligands bind in the pore. A mechanism describing catalysis is proposed that is consistent with the binding results.

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.