Abstract
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 kcat. However a third double mutant, K32M: 1+3, that places two mutations on the same half pore, enhances kcat 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the kcat/Km 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.
Highlights
R67 dihydrofolate reductase (R67 DHFR)1 is an R-plasmidencoded enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate
A single K32M:1 mutant was constructed3 where the mutation was placed in gene copy 1
Three critical observations from our asymmetric K32M mutations that provide further detail on R67 DHFR function are: 1) a non-productive binding mode has been uncovered in R67 DHFR as all three double mutants show similar kcat/Km values
Summary
R67 dihydrofolate reductase (R67 DHFR) 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. Binding of neither ligand can be optimized, and a “one-site-fits-both” approach is employed [3, 4] Another constraint arising from the symmetry is that addition of a mutation to the gene results in four mutations per single active site pore. Previous studies have identified Lys-32, Gln-67, Ile-68, and Tyr-69 as the most critical residues in binding and catalysis [4, 7,8,9].2 These residues were targeted for introduction of asymmetric muta-. Glu, the para-aminobenzoic acid-glutamate tail of dihydrofolate; MES, 4-morpholineethanesulfonic acid; Quad, the protein product of the quadruplicated R67 DHFR gene
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