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Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
Published on: March 20, 2021
Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1
Natalia Mast1, Andrew J Annalora, David T Lodowski
1Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.
The Journal of Biological Chemistry
|December 17, 2010
Summary
Mitochondrial cytochrome P450 11A1 (CYP11A1) crystal structure reveals how it binds cholesterol intermediates. This provides insights into steroid hormone synthesis and enzyme efficiency.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Mitochondrial cytochrome P450 11A1 (CYP11A1) is crucial for steroidogenesis, catalyzing cholesterol side-chain cleavage to produce pregnenolone.
- Pregnenolone is the precursor for all steroid hormones, synthesized through sequential monooxygenation reactions.
Purpose of the Study:
- To determine the crystal structure of CYP11A1 in complex with its first reaction intermediate, 22R-hydroxycholesterol (22HC).
- To elucidate the structural basis for CYP11A1's substrate specificity and catalytic efficiency.
Main Methods:
- X-ray crystallography was used to obtain the 2.5-Å crystal structure of the CYP11A1-22HC complex.
- Solution studies were employed to investigate the dynamics of 22HC within the enzyme.
Main Results:
- The crystal structure reveals a long, curved active site cavity where 22HC binds with its 22R-hydroxyl group near the heme iron.
- Ordered water molecules at the active site entrance facilitate "soft" recognition of the 22HC 3β-hydroxyl, suggesting intermediate shuttling.
- The structure highlights conserved motifs involved in mitochondrial P450 redox partner interactions.
Conclusions:
- The CYP11A1-22HC complex structure provides a molecular understanding of cholesterol side-chain cleavage.
- The findings suggest a dynamic mechanism involving intermediate shuttling for the multi-step catalytic process.
- This work offers insights into the structural basis of enzyme specificity and efficiency in steroid hormone biosynthesis.
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