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Interligand Overhauser effects in type II dihydrofolate reductase
1Laboratory of Structural Biology, MR-01, National Institute of Environmental and Health Sciences, Box 12233, Research Triangle Park, North Carolina 27709, USA.
Biochemistry
|April 4, 2001
Summary
Bacterial R67 dihydrofolate reductase (DHFR) forms a ternary complex with NADP(+) and folate. NMR studies reveal a unique ligand orientation within the enzyme
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- R67 dihydrofolate reductase (DHFR) is a type II enzyme conferring bacterial resistance to trimethoprim.
- Type II DHFRs are structurally distinct from chromosomal DHFRs and form homotetramers with a single active site pore.
- The precise structure of the ternary complex involving substrate and cofactor for type II DHFRs remains unclear.
Purpose of the Study:
- To elucidate the structure of the ternary complex formed by R67 DHFR, NADP(+), and folate using transferred NOE and ILOE techniques.
- To investigate the conformational states of NADP(+) and folate within the active site.
- To compare the ternary complex structure with that of chromosomal DHFRs and theoretical models.
Main Methods:
- Utilized transferred NOE and interligand NOE (ILOE) spectroscopy to analyze ligand interactions within the R67 DHFR ternary complex.
- Studied the binding of NADP(+) and folate, as well as a folate analogue (DMDDF), to R67 DHFR.
- Employed higher enzyme concentrations and temperatures for studies with the DMDDF analogue due to poorer exchange characteristics.
Main Results:
- Determined that NADP(+) adopts a syn conformation for the ribonicotinamide bond and an anti conformation for the adenosine moiety.
- Observed significant ILOE peaks between NADP(+) and folate, indicating a specific relative orientation within the active site pore.
- The orientation of NADP(+) and folate/DMDDF differs from chromosomal DHFRs, with ligands extending in opposite directions parallel to the pore axis and ring systems stacked centrally.
Conclusions:
- The ternary complex of R67 DHFR exhibits a unique structure where NADP(+) and folate/DMDDF ligands are oriented in opposite directions within the active site pore.
- This structure is consistent with theoretical calculations of the dihydrofolate-NADPH transition state.
- The findings provide crucial insights into the mechanism of type II DHFRs and their interaction with inhibitors.