R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.

R67 dihydrofolate reductase (DHFR) is an enzyme, encoded by an R-plasmid, that confers resistance to the antibacterial agent trimethoprim. This homotetramer possesses a single active site pore and exact 222 symmetry. The symmetry imposes constraints on the ability of the enzyme to optimize binding of the substrate, dihydrofolate (DHF), and the cofactor, NADPH, resulting in a "one site fits both ligands" approach. This approach allows formation of either a NADPH.NADPH, dihydrofolate.dihydrofolate, or NADPH.dihydrofolate complex. The first two complexes are nonproductive, while the third is the productive catalytic species. To break the symmetry of the active site, a tandem array of four R67 DHFR genes has been linked in frame, allowing individual manipulation of each gene copy. Various numbers and combinations of asymmetric Q67H mutations have been engineered into the tandem gene array. The Q67H mutation was chosen for investigation as it was previously found to tighten binding to both dihydrofolate and NADPH by approximately 100-fold in homotetrameric R67 DHFR [Park, H., Bradrick, T. D., and Howell, E. E. (1997) Protein Eng. 10, 1415-1424]. Nonadditive effects on ligand binding are observed when one to four mutations are inserted, indicating either conformational changes in the protein or different cooperativity patterns in the ligand-ligand interactions. From steady state kinetics, addition of Q67H mutations does not drastically affect formation of the NADPH.dihydrofolate complex; however, a large energy difference between the productive and nonproductive complexes is no longer maintained. A role for Q67 in discriminating between these various states is proposed. Since theories of protein evolution suggest gene duplication followed by accumulation of mutations can lead to divergence of activity, this study is a first step toward asking if introduction of asymmetric mutations in the quadrupled R67 DHFR gene can lead to optimization of ligand binding sites.

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569 -10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1؉2 and K32M: 1؉4, which place the mutations on opposite sides of the pore, reduce k cat . However a third double mutant, K32M: 1؉3, that places two mutations on the same half pore, enhances k cat 4-to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the k cat /K m values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1؉3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore. R67 dihydrofolate reductase (R67 DHFR)1 is an R-plasmidencoded enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. Its presence in bacteria confers resistance to the antibiotic, trimethoprim. This enzyme is not similar in sequence or structure to the chromosomally encoded DHFRs.R67 DHFR is a homotetramer, and the pore that traverses the length of the molecule is the active site. Surprisingly, the structure possesses 222 symmetry (1), which imposes numerous constraints on binding and catalysis. For example, the symmetry requires that for each binding site, there must be three additional, symmetry-related sites. However, solution studies find only two sites can be occupied simultaneously because of steric constraints. The possible binding combinations are two NADPH molecules, or two folate/DHF molecules, or one NADPH plus one folate/DHF molecule (2). Only the latter is productive. Thus binding of neither ligand can be optimized, and a "one-site-fits-both" approach is employed (3, 4). Another constraint arising from ...

R67 dihydrofolate reductase (DHFR) is a novel protein that possesses 222 symmetry. A single active site pore traverses the length of the homotetramer. Although the 222 symmetry implies that four symmetry-related binding sites should exist for each substrate as well as each cofactor, isothermal titration calorimetry (ITC) studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate with one cofactor. The latter is the productive ternary complex. To evaluate the roles of A36, Y46, T51, G64, and V66 residues in binding and catalysis, a site-directed mutagenesis approach was employed. One mutation per gene produces four mutations per active site pore, which often result in large cumulative effects. Conservative mutations at these positions either eliminate the ability of the gene to confer trimethoprim resistance or have no effect on catalysis. This result, in conjunction with previous mutagenesis studies on K32, K33, S65, Q67, I68, and Y69 [Strader, M. B., et al. (2001) Biochemistry 40, 11344-11352; Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Park, H., et al. (1997) Protein Eng. 10, 1415-1424], allows mapping of the active site surface. Residues for which conservative mutations have large effects on binding and catalysis include K32, Q67, I68, and Y69. These residues form a stripe that establishes the ligand binding surface. Residues that accommodate conservative mutations that do not greatly affect catalysis include K33, Y46, T51, S65, and V66. Isothermal titration calorimetry studies were also conducted on many of the mutants described above to determine the enthalpy of folate binding to the R67 DHFR.NADPH complex. A linear correlation between this DeltaH value and log k(cat)/K(m) is observed. Since structural tightness appears to be correlated with the exothermicity of the binding interaction, this leads to the hypothesis that enthalpy-driven formation of the ternary complex in these R67 DHFR variants plays a strong role in catalysis. Use of the alternate cofactor, NADH, extends this correlation, indicating preorganization of the ternary complex determines the efficiency of the reaction. This hypothesis is consistent with data suggesting R67 DHFR uses an endo transition state (where the nicotinamide ring of cofactor overlaps the more bulky side of the substrate's pteridine ring).

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