Dual-Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Synthesis

Background:

• Cytochrome P450 enzymes are crucial catalysts but often require expensive cofactors like NAD(P)H.
• Activating P450s for direct use with hydrogen peroxide (H2O2) has been a significant challenge.
• Developing cofactor-independent P450 systems is key for efficient biocatalysis.

Purpose of the Study:

• To engineer a novel H2O2-dependent cytochrome P450BM3 system.
• To utilize dual-functional small molecules (DFSMs) for enzyme activation and catalysis.
• To demonstrate enhanced peroxygenase activity for non-native substrates.

Main Methods:

• Design and synthesis of DFSMs, specifically N-(ω-imidazolyl fatty acyl)-l-amino acids.
• Characterization of the P450BM3-DFSM system for H2O2 activation.
• Assay of catalytic activity for epoxidation, sulfoxidation, and hydroxylation reactions.

Main Results:

• The developed system efficiently catalyzes monooxygenation of non-native substrates using H2O2.
• DFSMs anchor to the enzyme, with the imidazolyl group activating H2O2 via acid-base catalysis.
• Achieved superior peroxygenase activity for styrene epoxidation, thioanisole sulfoxidation, and ethylbenzene hydroxylation compared to previous P450-H2O2 systems.
• Demonstrated the first successful activation of H2O2-inert P450s using an exogenous small molecule.

Conclusions:

• This work presents a unique strategy for creating H2O2-dependent P450 systems.
• The approach circumvents the need for NAD(P)H and associated electron transport systems, reducing costs.
• This method offers a promising avenue for expanding the application of P450s in organic synthesis through direct chemical intervention.