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.
Highlights
Edited by Ruma BanerjeeP450 family 4 fatty acid -hydroxylases preferentially oxygenate primary C–H bonds over adjacent, energetically favored secondary C–H bonds, but the mechanism explaining this intriguing preference is unclear
Summary—The structure of rabbit P450 4B1 is a prototype for a group of P450 enzymes with covalently bound heme prosthetic groups
These include family 4 enzymes annotated in insects as well as annotated and unannotated P450s in other vertebrate and invertebrate animal species that have amino acid sequences with the conserved I-helix motif seen in P450 4B1 with a glutamic acid rather than the more common alanine or glycine present in other P450s
Summary
P450 family 4 fatty acid -hydroxylases preferentially oxygenate primary C–H bonds over adjacent, energetically favored secondary C–H bonds, but the mechanism explaining this intriguing preference is unclear. Fatty acid -hydroxylation reflects the addition of oxygen to a primary C–H bond of the terminal carbon of the fatty acid This reaction is generally considered to be the most difficult reaction catalyzed by cytochrome P450 monooxygenases because the enzyme needs to exclude access of neighboring and more reactive secondary C–H bonds to the iron-bound reactive oxygen intermediate. As the strength of primary C–H bonds is greater than that of secondary C–H bonds, oxygen addition at the -1 or -2 positions of fatty acids is typically seen for less specialized enzymes [15,16,17] These considerations suggest that the -hydroxylases have structural features that favor the approach to the reactive iron oxo species of the -carbon relative to the -1 carbon [18]. Comparisons of the amino acid sequence of P450 4B1 with other family 4 -hydroxylases indicate that amino acids that restrain octane for -hydroxylation are highly conserved in these enzymes
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