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Background: CYP11B1 and CYP11B2 are key enzymes in corticosteroid biosynthesis. The metabolites produced by these enzymes regulate essential physiological processes, including immune function and stress responses. Because the two enzymes share approximately 93% amino acid sequence identity, achieving selective inhibition of one over the other is challenging. Therefore, the objective of this study is to clearly define the functional and structural differences between CYP11B1 and CYP11B2.
Methods: Both proteins were expressed in Escherichia coli, and binding assays were performed using 11-deoxycortisol and 11-deoxycorticosterone as substrates. To obtain steady-state kinetic data, each enzyme was incubated with its respective substrate, and product formation was quantified by UPLC–MS/MS to determine enzymatic activity.
Results: Accurate Kd determination for CYP11B1 was not achievable due to poor binding assay performance. For CYP11B2, the Kd value was 4.8 ± 0.31 µM with 11-deoxycortisol and 2.08 ± 0.24 µM with 11-deoxycorticosterone (11-DOC). The catalytic efficiency (kcat/Km) of CYP11B1 was 0.26 ± 0.02 µM⁻¹·min⁻¹ with 11-deoxycortisol and 1.45 ± 0.4 µM⁻¹·min⁻¹ with 11-DOC, whereas CYP11B2 exhibited values of 1.62 ± 0.2 µM⁻¹·min⁻¹ and 0.28 ± 0.08 µM⁻¹·min⁻¹ with 11-deoxycortisol and 11-DOC, respectively.
Conclusion: The two enzymes exhibited differences in both substrate binding and catalytic activity, and further studies are planned to investigate the structural or mechanistic factors underlying these differences.
Acknowledgements: Supported by National Research Foundation of Korea, grant No. RS-2025-25438309
