Converting cytochrome c into a DyP-like metalloenzyme

Area of Science:

• Biotechnology
• Environmental Science
• Enzyme Engineering

Background:

• Dye-decolorizing peroxidase (DyP) shows potential for environmental purification by degrading anthraquinone dyes.
• Previous attempts to engineer DyP for enhanced dye degradation faced challenges with heme incorporation in Escherichia coli.
• Cytochrome c (cyt c), with its covalently bound heme, was explored as a scaffold for artificial DyP design.

Purpose of the Study:

• To engineer an artificial peroxidase based on cytochrome c for efficient degradation of reactive blue 19 (RB19).
• To enhance the catalytic efficiency and substrate binding affinity of cytochrome c for dye decolorization.
• To investigate mutations improving hydrogen bonding and reactivity with H2O2 for artificial DyP development.

Main Methods:

• Constructed artificial peroxidases based on cytochrome c.
• Introduced mutations including Met80Ala/Val, Pro76Trp (P76W), and Gly29Asp (G29D).
• Evaluated dye-decolorizing activity and catalytic efficiency (kcat/Km) of wild-type and mutant cytochrome c.

Main Results:

• Wild-type cytochrome c showed partial RB19 degradation, but less effective than Vibrio cholerae DyP at optimal pH.
• The P76W mutant exhibited a 3-fold increase in catalytic efficiency compared to wild-type cytochrome c.
• The G29D mutant demonstrated an approximately 80-fold enhancement in dye-decolorizing activity.

Conclusions:

• The G29D mutation significantly enhances the dye-decolorizing capability of cytochrome c-based artificial peroxidases.
• While the G29D mutant showed promise, heme degradation limited its application in Escherichia coli.
• This study provides valuable insights for designing novel artificial DyPs for bioremediation applications.