Speaker
Description
Background: Cytochrome P450 11A1 (CYP11A1) is a mitochondrial cytochrome P450 enzyme that catalyzes the conversion of cholesterol to pregnenolone, the first step in steroid hormone biosynthesis, thereby playing a critical role in regulating diverse physiological processes. CYP11A1 requires two redox partners, adrenodoxin reductase (AdR) and adrenodoxin (Adx), to receive electrons from NADPH. Although a shuttle model in which Adx dynamically transfers electrons between CYP11A1 and AdR has been proposed, quantitative experimental evidence for protein–protein interactions remains limited.
Methods and Results: In this study, human CYP11A1 was engineered to enhance expression in Escherichia coli by modifying its N-terminal sequence based on bovine CYP11A1. CYP11A1, bovine Adx, and bovine AdR were expressed and purified using Ni-NTA affinity chromatography. To quantify CYP11A1–Adx interaction, a cysteine-substituted CYP11A1 mutant was site-specifically labeled with Alexa Fluor 488, and fluorescence titration assays were performed. In addition, substrate-binding assays based on absolute absorbance spectroscopy were used to determine cholesterol-binding affinity for wild-type CYP11A1 and clinically reported mutants.
Conclusions: This work establishes experimental platforms for analyzing protein–protein and protein–substrate interactions in the mitochondrial CYP11A1 system and provides a foundation for understanding the mechanistic impact of disease-associated mutations.
Acknowledgments:
Supported by the National Research Foundation of Korea (NRF) grant RS-2024-00335140
