A novel P450 enzyme assay utilizing an NADP-based biosensor.

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.

Engineering Compartmentalization and Cofactor Regulation for 10-Hydroxy-2-decenoic Acid Biosynthesis in .

10-Hydroxy-2-decenoic acid (10-HDA), a bioactive component of royal jelly, exhibits significant pharmacological value in various applications. To date, its biosynthesis has been reported exclusively in endotoxin-prone , which limits its biomedical utility. In this study, we successfully engineered the Generally Recognized As Safe (GRAS) yeast as a biosynthetic platform for producing 10-HDA, thereby addressing the limitations associated with systems.

Understanding Cytochrome P450 Enzyme Substrate Inhibition and Prospects for Elimination Strategies.

Cytochrome P450 (CYP450) enzymes, which are widely distributed and pivotal in various biochemical reactions, catalyze diverse processes such as hydroxylation, epoxidation, dehydrogenation, dealkylation, nitrification, and bond formation. These enzymes have been applied in drug metabolism, antibiotic production, bioremediation, and fine chemical synthesis. Recent research revealed that CYP450 catalytic kinetics deviated from the classic Michaelis-Menten model.