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Volume 271, Number 37, Issue of September 13, 1996 pp. 22663-22671
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Highly Stereoselective Oxidation of Polyunsaturated Fatty Acids by Cytochrome P450BM-3

(Received for publication, April 15, 1996, and in revised form, July 2, 1996)

Jorge H. Capdevila ^§ , Shozou Wei ^§ , Christian Helvig ^§ , John R. Falck ^¶ , Yuri Belosludtsev ^¶ , Gilles Truan , Sandra E. Graham-Lorence and Julian A. Peterson

From Departments of  Biochemistry and ^¶ Molecular Genetics, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75325-9038 and Departments of ^§ Medicine and  Biochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232

Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S),15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 ± 0.4 µmol/min/nmol at 30 °C, the fastest ever reported for an NADPH-dependent, P450-catalyzed reaction. Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstrating efficient coupling of electron transport to monooxygenation.

Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 ± 0.2 and 2.9 ± 0.1 µmol/min/nmol at 30 °C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regio- and stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid -1, -2, and -3 sp³ carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively.

Based on the above and on a model of arachidonic acid-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp³ carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).

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