Emerging Strategies for Modifying Cytochrome P450 Monooxygenases into Peroxizymes

Area of Science:

• Biocatalysis and Synthetic Chemistry
• Enzyme Engineering
• Oxidation Reactions

Background:

• Cytochrome P450 monooxygenases are powerful oxidizing enzymes with significant potential in synthetic chemistry and biology.
• Their practical application is limited by reliance on expensive nicotinamide cofactors (NAD(P)H) and redox partner proteins.
• Utilizing low-cost hydrogen peroxide (H2O2) directly is desirable, but the peroxide shunt pathway is often inefficient.

Purpose of the Study:

• To develop strategies for modifying P450 monooxygenases into efficient peroxizymes (peroxygenase and peroxidase) for biocatalysis.
• To explore the catalytic applications of engineered P450 peroxizymes in challenging selective C-H oxidation, oxygenation, and oxyfunctionalization reactions.
• To overcome limitations of traditional P450 catalysis by enabling direct use of H2O2.

Main Methods:

• Development of a dual-functional small molecule (DFSM) strategy to transform P450BM3 into a peroxygenase, facilitating heterolytic cleavage of H2O2.
• Mechanism-guided protein engineering of redox-sensitive residues to switch DFSM-facilitated P450BM3 peroxygenase to an efficient peroxidase.
• Implementation of an H2O2 tunnel engineering strategy to enable peroxygenase activity in various P450 monooxygenases.

Main Results:

• DFSM strategy enabled P450BM3 peroxygenase for challenging selective C-H oxidations, including alkane hydroxylation, aryl ether O-demethylation, styrene epoxidation, and alkylbenzene hydroxylation.
• Engineered P450 peroxidase facilitated direct nitration of unsaturated hydrocarbons (phenols, aromatic amines, styrene derivatives), achieving the first direct biological nitration of olefins.
• H2O2 tunnel engineering provided a general approach for creating P450 peroxygenases across different P450s.

Conclusions:

• Emerging strategies, including DFSM, redox-sensitive residue engineering, and H2O2 tunnel engineering, enable practical P450 peroxizyme biocatalysts.
• These engineered P450s expand the scope of P450 chemistry and catalysis, offering solutions for challenging selective oxidations.
• The developed strategies show potential for broad application to other P450 enzymes, advancing synthetic chemistry and biocatalysis.