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Updated: Sep 15, 2025
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
A novel P450 enzyme assay utilizing an NADP+-based biosensor
Sifan Shangguan1,2, Taichang Wang1,2, Di Zhao1,2
1State Key Laboratory of Green Papermaking and Resource Recycling, Qilu University of Technology, Jinan, 250353, Shandong, Republic of China.
Purpose:
High-throughput screening methods for cytochrome P450 enzymes (P450s), such as colorimetric, mass spectrometric, and fluorescence-based assays, often face limitations in throughput, real-time monitoring, and versatility.
Methods:
To address these challenges, we developed a novel biosensor leveraging glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation for real-time monitoring of intracellular NADP+ levels, enabling P450 activity detection. The sensor was applied to monitor P450 activity by tracking intracellular NADP+ dynamics, as P450s catalyze diverse substrate reactions and convert NADPH to NADP+ via their electron transport system. To enhance detection precision, intracellular NADP+ synthesis was reduced by knocking down NADPH-dependent aldehyde reductase (YqhD), minimizing background fluorescence interference.
Results:
The sensor exhibited a linear NADP+ detection range of 1 μM to 10 mM, suitable for P450 assays. The sensor's performance was validated by comparing P450 activities in engineered strains with traditional gas chromatography.
Conclusion:
The developed biosensor demonstrates its potential as a robust, real-time screening tool for P450 enzyme studies.
