The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation*♦
- From the ‡Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037 and
- the §Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195
- ↵1 To whom correspondence should be addressed: The Scripps Research Institute, MEM-255, 10550 North Torrey Pines Rd., La Jolla, CA 92037. E-mail: johnson{at}scripps.edu.
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Edited by Ruma Banerjee
Abstract
P450 family 4 fatty acid ω-hydroxylases preferentially oxygenate primary C–H bonds over adjacent, energetically favored secondary C–H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for ω-hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C–H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for ω-hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for ω-hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane ω-hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for ω-hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for ω-hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C–H bonds.
- cytochrome P450
- enzyme structure
- fatty acid metabolism
- heme
- membrane protein
- X-ray crystallography
- xenobiotic
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM031001 (to E. F. J.), GM007750 (to B. R. B.), and GM049054 (to A. E. R.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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↵♦ This article was selected as one of our Editors' Picks.
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This article contains supplemental Fig. S1 and supplemental File P450–4A11-homology-model.pdb.
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The atomic coordinates and structure factors (code 5T6Q) have been deposited in the Protein Data Bank (http://wwpdb.org/).
- Received January 6, 2017.
- Revision received February 2, 2017.
- © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
