Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin

Area of Science:

• Biochemistry
• Protein Engineering
• Enzyme Catalysis

Background:

• Heme metalloproteins like myoglobin (Mb) are crucial biological catalysts.
• Unnatural amino acids offer novel functionalities when incorporated into protein scaffolds.
• The distal histidine in Mb plays a key role in modulating its enzymatic activity.

Purpose of the Study:

• To investigate the potential of incorporating unnatural amino acids into heme metalloproteins.
• To engineer a myoglobin mutant with enhanced peroxide activation capabilities.
• To explore the catalytic role of 3-amino-l-tyrosine (NH2Tyr) in the Mb active site.

Main Methods:

• Genetic engineering of myoglobin to replace distal histidine (H64) with NH2Tyr.
• Peroxide-shunt assays to measure oxidation rates of thioanisole and benzaldehyde.
• Kinetic, electrochemical, and Electron Paramagnetic Resonance (EPR) spectroscopy for characterization.

Main Results:

• The H64NH2Tyr Mb mutant exhibited significantly increased turnover rates compared to wild-type Mb.
• A 9-fold increase in thioanisole sulfoxidation and an 81-fold increase in benzoic acid formation were observed.
• NH2Tyr in the distal active site was crucial for enhanced peroxide activation.

Conclusions:

• Unnatural amino acids can confer non-natural functions to protein scaffolds.
• The NH2Tyr residue in the engineered Mb mimics the function of the His/Arg pair in horseradish peroxidase.
• This study highlights a novel strategy for engineering enhanced enzymatic activity in heme proteins.