Tunnel engineering for modulating the substrate preference in cytochrome P450_BsβHI.

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Enzymes utilize internal active sites accessible via substrate tunnels.
• Tunnel engineering is a key strategy to enhance enzyme catalytic properties.
• P450BsβHI, a potential decarboxylase from Bacillus subtilis, exhibits limited activity on long-chain fatty acids.

Purpose of the Study:

• To engineer the substrate tunnel of P450BsβHI to improve its decarboxylase activity.
• To modulate the substrate preference of P450BsβHI for long-chain fatty acids.

Main Methods:

• Site-directed mutagenesis was employed to create variants of P450BsβHI.
• Tunnel engineering strategies were applied to modify the enzyme's substrate access pathway.

Main Results:

• The BsβHI-F79A variant showed a 15.2-fold increase in palmitic acid conversion.
• The BsβHI-F173V variant demonstrated a 3.9-fold improvement in pentadecanoic acid conversion.
• Specific mutations successfully altered substrate preference and enhanced decarboxylation efficiency.

Conclusions:

• Tunnel engineering is an effective strategy for optimizing enzyme function.
• This approach can be used to tailor enzymes for specific substrates, such as long-chain fatty acids.
• The study provides a framework for enhancing decarboxylase activity through protein engineering.