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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.

Biotechnology Letters
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Summary

A novel biosensor enables real-time monitoring of cytochrome P450 (P450) enzyme activity by tracking intracellular NADP+ levels. This tool offers a robust solution for high-throughput screening of P450s.

Keywords:
CRISPR/Cas9HemeNADP + sensorP450 enzyme detection

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Traditional high-throughput screening methods for cytochrome P450 enzymes (P450s) have limitations in throughput, real-time monitoring, and versatility.
  • Existing assays like colorimetric, mass spectrometric, and fluorescence-based methods do not fully meet the demands of modern biochemical research.

Purpose of the Study:

  • To develop a novel biosensor for real-time monitoring of intracellular NADP+ levels to enable P450 activity detection.
  • To overcome the limitations of current P450 screening techniques by providing a more versatile and precise method.

Main Methods:

  • Developed a biosensor using glucose-6-phosphate dehydrogenase and Bimolecular Fluorescence Complementation to monitor intracellular NADP+ dynamics.
  • Applied the sensor to track P450 activity by measuring the conversion of NADPH to NADP+.
  • Reduced background fluorescence by knocking down NADPH-dependent aldehyde reductase (YqhD) to enhance detection precision.

Main Results:

  • The biosensor demonstrated a linear NADP+ detection range from 1 μM to 10 mM, suitable for P450 assays.
  • Validated sensor performance by comparing P450 activities in engineered strains with traditional gas chromatography.
  • Successfully monitored intracellular NADP+ dynamics to infer P450 enzyme activity.

Conclusions:

  • The developed biosensor is a robust tool for real-time screening of P450 enzyme activity.
  • This novel approach holds significant potential for advancing P450 research and drug discovery.