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R plasmid dihydrofolate reductase with subunit structure.

S L Smith, D Stone, P Novak

    The Journal of Biological Chemistry
    |July 25, 1979
    PubMed
    Summary
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    Researchers purified dihydrofolate reductase (DHFR) from trimethoprim-resistant Escherichia coli. The enzyme is a novel tetramer, distinct from other bacterial and vertebrate DHFR enzymes.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Microbiology

    Background:

    • Dihydrofolate reductase (DHFR) is a crucial enzyme in folate metabolism.
    • Antibiotic resistance, particularly to trimethoprim, is a significant public health concern.
    • Understanding the structure of resistant enzymes can inform drug development.

    Purpose of the Study:

    • To purify and characterize dihydrofolate reductase (DHFR) from a trimethoprim-resistant Escherichia coli strain carrying a type II plasmid.
    • To investigate the quaternary structure and subunit composition of this specific DHFR enzyme.
    • To compare its properties with known bacterial and vertebrate DHFR enzymes.

    Main Methods:

    • Purification of DHFR using gel filtration, DEAE-Sephacel chromatography, and hydrophobic chromatography.

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  • Analysis of enzyme purity and molecular weight via polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE (SDS-PAGE).
  • Determination of molecular weight using gel filtration and Ferguson analysis.
  • Partial amino acid sequencing.

    • Main Results:

    • DHFR was purified 40-fold to homogeneity with a specific activity of 1.0 unit/mg.
    • Native PAGE and gel filtration indicated a molecular weight of 36,000 Da.
    • SDS-PAGE revealed a subunit molecular weight of 8,500 Da.
    • These findings suggest the enzyme is a tetramer of four identical subunits.
    • Partial sequencing showed no subunit heterogeneity and no clear homology to other reported reductase sequences.

    Conclusions:

    • The type II R plasmid-encoded DHFR from trimethoprim-resistant E. coli possesses a unique tetrameric structure.
    • This quaternary structure differs from previously characterized bacterial and vertebrate DHFR enzymes.
    • The enzyme's distinct structure may have implications for its interaction with trimethoprim and warrants further investigation.