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Related Experiment Videos

Interligand Overhauser effects in type II dihydrofolate reductase.

D Li1, L A Levy, S A Gabel

  • 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
PubMed
Summary

Bacterial R67 dihydrofolate reductase (DHFR) forms a ternary complex with NADP(+) and folate. NMR studies reveal a unique ligand orientation within the enzyme

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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.

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  • 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.