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| Main Authors: | , , , , , , , , |
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| Format: | Artículo científico |
| Language: | en |
| Published: |
Synthetic and systems biotechnology
2026
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| Online Access: | https://pubmed.ncbi.nlm.nih.gov/41756023/ |
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Table of Contents:
- P450 engineering via structure-guided rational design achieves high C21-selectivity and bioconversion in steroid biosynthesis. Yang, Jian Li, Rong Gao, Qilin Ma, Li Wang, Qiang Ma, Guiying Fan, Yikang Li, Shengying Xu, Lian-Hua C21-hydroxylation is a crucial step in the synthesis of corticosteroids. For instance, C21-hydroxylase catalyzes the conversion of progesterone (PRO) into 11-deoxycorticosterone (DOC, the precursor of cortisol and aldosterone). Through structure-guided rational design combined with focused rational iterative site-directed mutagenesis (FRISM), we engineered the wild-type CYP154C5 from to obtain the highly efficient variant M6a (F92A/V291L/L294I/Q239K/F180W/Q398M). This variant demonstrated a complete regioselectivity shift from C16α to C21 hydroxylation (98% selectivity) of PRO with high catalytic efficiency (>99% conversion). To further enhance catalytic performance, we engineered a redox fusion variant (M6a-RhFRED L3) that demonstrated the highest catalytic activity, achieving a 1.43-fold improvement compared to the M6a-RhFRED. Additionally, M6a-RhFRED L3 catalyzed the C21-hydroxylation of three steroid analogs (dydrogesterone, 16-dehydroprogesterone, and pregna-4,6-diene-3,20-dione), with selectivity reaching 98% and conversion rates exceeding 60%. Molecular docking and molecular dynamics (MD) simulations revealed that the PRO substrate undergoes conformational rearrangement in the M6a active site. This structural reorganization underscores the critical role of key residues in modulating regioselectivity, particularly the shift from C16α to C21 hydroxylation. This study not only provides an efficient biocatalyst for steroids C21-hydroxylation but also offers valuable insight for the rational engineering of the P450 enzymes.