The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3.
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 micromol/min/nmol at 30 degrees 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 micromol/min/nmol at 30 degrees 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 omega-1, omega-2, and omega-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).
Capdevila
J H
JH
Department of Biochemistry, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
Wei
S
S
Helvig
C
C
Falck
J R
JR
Belosludtsev
Y
Y
Truan
G
G
Graham-Lorence
S E
SE
Peterson
J A
JA
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008958:Models, Molecular; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D010957:Plasmids / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013237:Stereoisomerism
D001426:Bacterial Proteins; D005231:Fatty Acids, Unsaturated; D006893:Hydroxyeicosatetraenoic Acids; C061444:18-hydroxy-5,8,11,14-eicosatetraenoic acid; D016718:Arachidonic Acid; D009249:NADP; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D015126:8,11,14-Eicosatrienoic Acid