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Updated: Feb 11, 2026

Synthesis of Keratin-based Nanofiber for Biomedical Engineering
Published on: February 7, 2016
Structure-Guided Engineering of KshA Yields a High-Performance 9α-Hydroxylase for Synthesis of 9α-OH-AD
Yanmei Dai1,2, Dongchang Sun2, Qingyu Zhang1
1College of Pharmaceutical Science, Zhejiang Key Laboratory of Green Manufacturing Technology for Chemical Drugs, Zhejiang University of Technology, Hangzhou 310014, China.
Abstract:
9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) is a crucial steroid pharmaceutical intermediate synthesized from androst-4-ene-3,17-dione (4-AD) via catalysis by 9α-hydroxylase (KSH), which comprises KshA and KshB subunits. KshB supplies electrons to activate the [2Fe-2S] center in KshA, enabling 4-AD hydroxylation. However, KSH's stability and activity limit industrial 9α-OH-AD production. This study identified KshA as a key bottleneck and revealed four mutation hotspots through structure-guided mutagenesis. Ultimately, a triple mutant KshAL263A/G321N/D325 K with superior performance was obtained. This variant KSH exhibited a 10.3-fold increase in activity and a 5.04-fold improvement in kcat/Km in comparison to the wild-type. Molecular simulations indicated enhanced structural stability and substrate accessibility. The engineered strain Escherichia coli BL21-pET28a+-KshAL263A/G321N/D325 K/pETDuet-1-KshB-FDH achieved 119.8 mM 9α-OH-AD from 4-AD in fed-batch transformation, demonstrating a high-performance KSH variant for efficient industrial production.
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