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Related Experiment Videos

Changing the substrate specificity of cytochrome c peroxidase using directed evolution.

A Iffland1, S Gendreizig, P Tafelmeyer

  • 1Institut de Chimie Organique, Université de Lausanne, CH-1015 Lausanne, Switzerland.

Biochemical and Biophysical Research Communications
|August 4, 2001
PubMed
Summary

Directed evolution of yeast cytochrome c peroxidase (CCP) created mutants with over 20-fold enhanced activity and specificity for the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

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Area of Science:

  • Enzymology
  • Protein Engineering
  • Biocatalysis

Background:

  • Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae is a key enzyme in cellular defense.
  • Understanding CCP's substrate specificity is crucial for enzyme engineering and biocatalysis.
  • Directed evolution offers a powerful approach to modify enzyme properties.

Purpose of the Study:

  • To engineer Saccharomyces cerevisiae cytochrome c peroxidase (CCP) for enhanced activity against 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).
  • To investigate the structural basis for altered substrate specificity in CCP mutants.
  • To improve the expression and selection of active CCP variants.

Main Methods:

  • Directed molecular evolution, including DNA shuffling and saturation mutagenesis.

Related Experiment Videos

  • Enzyme activity assays using 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol.
  • Site-directed mutagenesis to identify key residues responsible for activity changes.
  • Protein expression in Escherichia coli and colony screening.

    • Main Results:

    • Isolated CCP mutants exhibited over 20-fold increased activity and 70-fold increased specificity toward ABTS compared to the natural substrate.
    • Activities against guaiacol remained largely unaffected, indicating selective enhancement.
    • Mutations at Asp224 and Asp217 were identified as critical for the observed increase in ABTS activity.
    • Mutations at Asp224 also enhanced the expression of active holoenzyme in Escherichia coli.

    Conclusions:

    • Directed evolution successfully generated CCP mutants with significantly improved catalytic performance for ABTS.
    • The identified mutations, particularly at Asp224 and Asp217, highlight novel regions influencing substrate specificity.
    • Enhanced holoenzyme expression facilitates the selection of beneficial mutations, aiding protein engineering efforts.