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A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site
N Narayana1, D A Matthews, E E Howell
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093, USA.
Abstract:
Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 M(r) homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 A resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 A in length, passing through the middle of the molecule. Two folate molecules bind asymmetrically within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.
Insights
High-level trimethoprim resistance in bacteria is linked to R67 plasmid-encoded dihydrofolate reductase (R67 DHFR). Its unique homotetrameric structure, unrelated to chromosomal forms, features a central pore where folate binds, revealing an unusual active site.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Bacteria can develop high-level resistance to the antibiotic trimethoprim.
- This resistance is often mediated by plasmid-encoded dihydrofolate reductase (R67 DHFR).
- Native R67 DHFR is a homotetramer that can exist in an equilibrium with an inactive dimeric form.
Purpose of the Study:
- To determine the three-dimensional structure of native R67 DHFR.
- To elucidate the structural basis for trimethoprim resistance conferred by R67 DHFR.
- To understand the mechanism of substrate binding in R67 DHFR.
Main Methods:
- X-ray crystallography was used to solve the structure of native R67 DHFR.
- The structure was determined at a resolution of 1.7 Å.
- Folate binding within the enzyme structure was analyzed.
Main Results:
- The structure of native R67 DHFR was solved, revealing a homotetrameric quaternary structure.
- R67 DHFR's structure is distinct and unrelated to that of chromosomal DHFR enzymes.
- A unique 25 Å pore runs through the center of the homotetrameric R67 DHFR.
- Two folate molecules were observed to bind asymmetrically within this central pore.
Conclusions:
- The unique structure of R67 DHFR, including its central pore, is responsible for high-level trimethoprim resistance.
- The active site of R67 DHFR is formed by contributions from all four subunits, utilizing symmetry-related binding surfaces.
- This asymmetric binding of folate within the pore suggests a novel mechanism of enzyme activity and inhibition.