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CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering*
Paula Bracco1, Hein J Wijma2, Bastian Nicolai1
1Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
Chembiochem : a European Journal of Chemical Biology
|November 4, 2020
Summary
Protein engineering of CYP154C5 (a P450 monooxygenase) altered its steroid hydroxylation regioselectivity. Mutant F92A produced 21-hydroxylated progesterone, offering insights into enzyme structure-function relationships.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- CYP154C5 is a P450 monooxygenase from Nocardia farcinica.
- It exhibits high regio- and stereoselectivity for 16α-hydroxylation of steroids.
Purpose of the Study:
- To engineer CYP154C5 for altered steroid hydroxylation regioselectivity.
- To investigate the structural basis for changes in enzyme activity and substrate binding.
Main Methods:
- Protein engineering of CYP154C5 based on crystal structure.
- Steroid hydroxylation assays with wild-type and mutant enzymes.
- Molecular Dynamics (MD) simulations to analyze enzyme-substrate interactions and active site dynamics.
Main Results:
- Mutant CYP154C5 F92A achieved altered regioselectivity, producing 21-hydroxylated progesterone (11-deoxycorticosterone) alongside 16α-hydroxylation.
- MD simulations indicated an alternative steroid binding mode in the F92A mutant active site.
- MD simulations suggested increased uncoupling in the F92A mutant due to water entry.
- Wild-type CYP154C5 exclusively performed 15α-hydroxylation on 5α-androstan-3-one.
Conclusions:
- Protein engineering can successfully modify the regioselectivity of CYP154C5 for steroid hydroxylation.
- Enzyme structure-function relationships were elucidated through mutant analysis and molecular simulations.
- Understanding active site dynamics and substrate binding is crucial for designing P450 enzymes with specific catalytic activities.
Keywords:
biocatalysiscytochrome P450 monooxygenaseprotein engineeringregioselectivitysteroid hydroxylationMore Related Videos
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