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

• Published in: The Journal of biological chemistry 1996-09, Vol.271 (37), p.22663-22671
• Main Authors: Capdevila, J H, Wei, S, Helvig, C, Falck, J R, Belosludtsev, Y, Truan, G, Graham-Lorence, S E, Peterson, J A
• Format: Article
• Language: English
• Subjects: 8,11,14-Eicosatrienoic Acid - analogs & derivatives; 8,11,14-Eicosatrienoic Acid - metabolism; Arachidonic Acid - metabolism; Bacillus megaterium; Bacterial Proteins; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System - metabolism; Fatty Acids, Unsaturated - metabolism; Hydroxyeicosatetraenoic Acids - metabolism; Mass Spectrometry; Mixed Function Oxygenases - metabolism; Models, Molecular; NADP - metabolism; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Plasmids - metabolism; Spectrophotometry, Atomic; Stereoisomerism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.37.22663

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 ω-1, ω-2, and ω-3 sp 3 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 sp 3 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).