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Related Experiment Videos

Circularly permuted beta-lactamase from Staphylococcus aureus PC1

U Pieper1, K Hayakawa, Z Li

  • 1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.

Biochemistry
|July 22, 1997
PubMed
Summary

Altering enzyme flexibility impacts beta-lactamase activity and stability. Circularly permuted mutants revealed how domain interface flexibility affects enzymatic function and protein structure.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Beta-lactamase enzymes from Staphylococcus aureus PC1 are crucial for antibiotic resistance.
  • Enzyme activity is often influenced by the flexibility and structural dynamics of its domains.
  • Understanding domain flexibility is key to enzyme engineering and drug development.

Purpose of the Study:

  • To investigate the role of domain flexibility in the enzymatic activity of Staphylococcus aureus PC1 beta-lactamase.
  • To explore how altering protein structure through circular permutation affects enzyme function and stability.
  • To analyze the impact of introducing flexibility at the domain interface on beta-lactamase activity.

Main Methods:

  • Production and characterization of two circularly permuted beta-lactamase mutants (cp254 and cp228).

Related Experiment Videos

  • Determination of crystal structure for cp254 at 1.8 A resolution.
  • Enzymatic activity assays using a panel of beta-lactam antibiotics.
  • Circular dichroism and thermal denaturation experiments to assess protein structure and stability.

    • Main Results:

    • Circularly permuted mutant cp254 exhibited similar structure and activity to wild-type beta-lactamase.
    • Mutant cp228, with enhanced flexibility at the domain interface, showed significantly reduced overall enzymatic activity.
    • Cp228 displayed a 10-fold increase in hydrolysis rate for cefotaxime, a third-generation cephalosporin.
    • Both mutants showed altered thermal stability compared to the wild-type enzyme, with cp228 being substantially less stable.

    Conclusions:

    • Introducing flexibility at the domain interface of beta-lactamase profoundly impacts enzyme activity and protein stability.
    • Specific alterations in flexibility can lead to both decreased general activity and enhanced hydrolysis of particular substrates.
    • Enzyme engineering through circular permutation offers insights into structure-function relationships and potential for targeted modifications.