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Improved 2α-Hydroxylation Efficiency of Steroids by CYP154C2 Using Structure-Guided Rational Design
Qilin Gao1, Bingbing Ma2, Qianwen Wang3
1College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou, China.
Applied and Environmental Microbiology
|February 27, 2023
Summary
Engineered Cytochrome P450 enzymes (CYP154C2) show significantly enhanced 2α-hydroxylation of steroids like androstenedione and testosterone. Structure-guided mutations improved conversion efficiency and substrate binding, paving the way for steroid drug biosynthesis.
Area of Science:
- Biocatalysis and enzyme engineering
- Structural biology and protein engineering
- Biotechnology and industrial microbiology
Background:
- Cytochrome P450 enzymes are valuable industrial biocatalysts due to their versatile C-H oxidation capabilities.
- Steroid hydroxylation is crucial for producing medically important steroid derivatives.
- Existing 2α-hydroxylation methods for steroids suffer from low efficiency and selectivity.
Purpose of the Study:
- To identify and engineer CYP154C2 from Streptomyces avermitilis for enhanced 2α-hydroxylation of androstenedione (ASD) and testosterone (TES).
- To elucidate the structural basis for CYP154C2's activity and selectivity through crystal structure analysis.
- To improve the conversion efficiency and selectivity of steroid 2α-hydroxylation using structure-guided mutagenesis.
Main Methods:
- In vitro conversion assays to evaluate enzyme activity.
- X-ray crystallography to determine the testosterone-bound structure of CYP154C2 at 1.42 Å resolution.
- Rational design and construction of eight CYP154C2 mutants (single, double, triple) based on structural insights.
- Biochemical characterization of wild-type and mutant enzymes, including kinetic analysis (kcat/Km) and substrate binding affinity measurements.
Main Results:
- The wild-type CYP154C2 exhibited 2α-hydroxylation activity toward ASD.
- Mutants L88F/M191F and M191F/V285L demonstrated significantly enhanced conversion rates (up to 46.5-fold for ASD) compared to the wild-type.
- Mutant L88F/M191F showed improved substrate binding affinity for both TES and ASD.
- Mutations, particularly L88F, were found to influence substrate binding orientation and selectivity, with L88F mutants producing 16α-hydroxylation products.
Conclusions:
- Structure-guided engineering of CYP154C2 efficiently enhanced the 2α-hydroxylation of steroids with high regio- and stereoselectivity.
- The L88 residue plays a critical role in determining substrate selectivity in the CYP154C subfamily.
- These findings provide a robust strategy and theoretical foundation for P450-mediated steroid 2α-hydroxylation, facilitating the development of steroid-based pharmaceuticals.
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