A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site

  title={A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site},
  author={Narendra Narayana and David A. Matthews and Elizabeth E. Howell and Nguyen-huu Xuong},
  journal={Nature Structural Biology},
Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 Mr homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 Å resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 Å in length, passing through the middle of the molecule. Two folate molecules bind… 

Mechanistic Studies of R67 Dihydrofolate Reductase

Results indicate protonated dihydrofolate (pKa = 2.59) is the productive substrate and that R67 DHFR does not possess a proton donor.

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

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.

Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure.

A tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF, and it was demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymosynthesis site had been introduced by these mutations.

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active‐site substitutions

The results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native‐like catalytic rate and thus ensure cellular proliferation, and suggests a high tolerance for active‐site substitutions.

Directed Evolution and Osmolyte Studies of R67 Dihydrofolate Reductase

An in vivo study was conducted monitoring the effect of the osmolyte sorbitol plus TMP on the growth of wild-type R67 DHFR and several mutants and the effects of heavily mutating a variant of this enzyme were investigated, supporting in vitro findings about the importance of available water to this enzyme.

Computational Development of Inhibitors of Plasmid-Borne Bacterial Dihydrofolate Reductase

The design of promising lead compounds to target R67 DHFR are described and it is shown that this candidate also binds strongly to the canonical prokaryotic dihydrofolate reductase and to human DHFR, and is therefore likely to be useful to the development of chemotherapeutic agents and of dual-acting antibiotics that target the two types of bacterial di hydrofolates.

Role of Lys-32 Residues in R67 Dihydrofolate Reductase Probed by Asymmetric Mutations*

The K32M:1+3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate, which arises from the 222 symmetry imposed on the single active site pore.

Crystal structure of a type II dihydrofolate reductase catalytic ternary complex.

The structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

Results suggest that multipe binding modes of the ligands are possible within R67 DHFR, a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim.



Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67.

A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase.

R plasmid dihydrofolate reductase with subunit structure.

Conformation of NADP+ bound to a type II dihydrofolate reductase.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs, and a derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized.

Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein.

Results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein, which may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.

Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase.

Although RBG200 DHFR is different both in sequence and in structure from known chromosomal enzymes, both enzymes catalyze identical hydrogen-transfer reactions, making it a member of the A-stereospecific class of dehydrogenases.

Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer<-->two-dimer equilibrium.

Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200-600-fold decrease in catalytic efficiency.

Alpha-pyridine nucleotides as substrates for a plasmid-specified dihydrofolate reductase.

  • S. SmithJ. Burchall
  • Biology, Chemistry
    Proceedings of the National Academy of Sciences of the United States of America
  • 1983
Data suggest that the enzyme from R plasmid R67 possesses a pyridine nucleotide binding site different from that of other dihydrofolate reductases and dehydrogenases.

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA, but the solution structure is determined, which is very similar to that found in eukaryotic Src homology-3 (SH3) domains.