R67 dihydrofolate reductase (DHFR) provides resistance to the antibacterial drug trimethoprim. This Rplasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of active, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018 -1025) indicates that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-directed mutagenesis of residues in the pore will produce four mutations/active site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the essential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDSpolyacrylamide gel electrophoresis and is monomeric under nondenaturing conditions as measured by sedimentation equilibrium experiments. The catalytic activity of quadruple R67 DHFR is decreased only slightly when compared with native R67 DHFR. Folding of quadruple R67 DHFR is completely reversible at pH 5. However, at pH 8, folding is not fully reversible; this is likely due to a competition between productive intramolecular versus nonproductive intermolecular domain association. The production of a fully active, monomeric R67 DHFR variant will enable the design of more meaningful site-directed mutants where single substitutions per active site pore can be generated.Recent protein engineering studies of oligomeric proteins have attempted to shift the assembly equilibrium toward the protomer by the introduction of destabilizing mutations at subunit interfaces (Refs. 1-3 and references therein). These studies allow the functional role of protein-protein interactions at subunit interfaces to be probed. This strategy assumes that most oligomeric proteins possessing n protomers possess n active sites and is less useful for enzymes that have active sites to which several subunits contribute.In contrast to the above approach that forms monomers by dissociation of the native oligomeric species, we have constructed an active, stable monomeric form of R67 dihydrofolate reductase (DHFR) 1 by association. This strategy involves the use of genetic engineering techniques to covalently link four protomers together. This approach is necessarily different, since R67 DHFR is a homotetramer with a single active site pore, and stabilization of the 78-amino acid monomer would yield only a partial active site.In R67 DHFR, a 222 symmetry element occurs at the center of the pore, and crystallography and binding studies indicate that two molecules of either substrate (dihydrofolate/folate) or cofactor (NADPH) can bind to both sides of the active site pore under binary complex conditions (4, 5). Under ternary complex conditions, a total of two ligands can be bound; the possibilities include two NADPH molecules, two folate molecules, or one NADPH and one folate bound on opposite sides of the pore. T...
