Artificial Self-Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase...

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Bacterial cytochrome P450 monooxygenases (P450s) typically require two auxiliary proteins for electron transfer.
• Existing artificial P450 systems with single auxiliary proteins have not reached optimal catalytic efficiency.

Purpose of the Study:

• To enhance the catalytic activity of Pseudomonas putida P450 (P450cam) by assembling it with multiple auxiliary proteins.
• To overcome catalytic bottlenecks in P450 systems through protein engineering.

Main Methods:

• Fusion of P450cam with varying numbers of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), to a heterotrimeric protein.
• Kinetic analysis to evaluate catalytic turnover and identify rate-limiting steps.

Main Results:

• Assembly with one PdX and one PdR showed catalytic cycles suspended due to slow PdX reduction.
• Increasing PdR molecules accelerated PdX reduction.
• Assembly with two PdXs and three PdRs achieved 92% of the maximum activity of free P450cam, eliminating waiting steps.

Conclusions:

• Optimizing the stoichiometry of auxiliary proteins is crucial for maximizing P450 catalytic efficiency.
• Engineered P450 systems with multiple auxiliary proteins show significant potential for in vitro biotechnological applications.