Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Chemical Biology

Background:

• Colorimetric assays are crucial for screening enzyme activity.
• P450 enzymes, particularly hybrid P450 BM3, are important for biocatalysis.
• Developing efficient screening methods for engineered enzymes is essential.

Purpose of the Study:

• To develop and validate a colorimetric assay for light-driven hybrid P450 BM3 enzymes.
• To investigate p-nitrophenoxy derivatives as chromogenic substrates for these enzymes.
• To enable high-throughput screening of next-generation P450 BM3 for C-H functionalization.

Main Methods:

• Incorporation of p-nitrophenoxy moiety into substrates.
• Utilizing Ru(II) photosensitizer for light-driven P450 BM3 activation.
• Adaptation of the assay to a 96-well plate format.

Main Results:

• Identified p-nitrophenoxy derivatives as effective chromogenic substrates.
• Demonstrated compatibility of substrates with the light-driven enzymatic approach.
• Validated the assay for screening O-demethylation activity.

Conclusions:

• The developed colorimetric assay is suitable for light-driven hybrid P450 BM3 enzymes.
• This assay facilitates rapid screening of enzyme variants for C-H bond functionalization.
• The 96-well plate format enables high-throughput screening of engineered biocatalysts.