REDESIGNING THE QUATERNARY STRUCTURE OF R67 DIHYDROFOLATE-REDUCTASE -CREATION OF AN ACTIVE MONOMER FROM A TETRAMERIC PROTEIN BY QUADRUPLICATION OF THE GENE

R67 dihydrofolate reductase (DHFR) provides resistance to the antibact erial drug trimethoprim. This R-plasmid-encoded enzyme does not share any homology with chromosomal DHFR. A recent crystal structure of acti ve, homotetrameric R67 DHFR (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025) indicate s that a single active site pore traverses the length of the molecule. Since the center of the pore possesses exact 222 symmetry, site-direc ted mutagenesis of residues in the pore will produce four mutations/ac tive site. To break this inevitable symmetry, four copies of the gene have been linked in frame to create an active monomer possessing the e ssential tertiary structure of native tetrameric R67 DHFR. The protein product, quadruple R67 DHFR, is 4 times the molecular mass of native R67 DHFR in SDS-polyacrylamide gel electrophoresis and is monomeric un der 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 quadrupl e 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 a ssociation. The production of a fully active, monomeric R67 DHFR varia nt will enable the design of more meaningful site directed mutants whe re single substitutions per active site pore can be generated.