The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3

Jorge H. Capdevila, Shozou Wei, Christian Helvig, J R Falck, Yuri Belosludtsev, Gilles Truan, Sandra E. Graham-Lorence, Julian A. Peterson

Research output: Contribution to journalArticle

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Abstract

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 regioand 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 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).

Original languageEnglish (US)
Pages (from-to)22663-22671
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number37
DOIs
StatePublished - 1996

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Cytochromes
Unsaturated Fatty Acids
Arachidonic Acid
Carbon
NADP
Oxidation
Acids
Eicosapentaenoic Acid
Hydroxylation
Hydroxyeicosatetraenoic Acids
Electron Transport
Heme
Oxidants
Epoxidation
Reactive Oxygen Species
Catalytic Domain
Fatty Acids
Metabolism
Oxygen
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Capdevila, J. H., Wei, S., Helvig, C., Falck, J. R., Belosludtsev, Y., Truan, G., ... Peterson, J. A. (1996). The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. Journal of Biological Chemistry, 271(37), 22663-22671. https://doi.org/10.1074/jbc.271.37.22663

The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. / Capdevila, Jorge H.; Wei, Shozou; Helvig, Christian; Falck, J R; Belosludtsev, Yuri; Truan, Gilles; Graham-Lorence, Sandra E.; Peterson, Julian A.

In: Journal of Biological Chemistry, Vol. 271, No. 37, 1996, p. 22663-22671.

Research output: Contribution to journalArticle

Capdevila, JH, Wei, S, Helvig, C, Falck, JR, Belosludtsev, Y, Truan, G, Graham-Lorence, SE & Peterson, JA 1996, 'The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3', Journal of Biological Chemistry, vol. 271, no. 37, pp. 22663-22671. https://doi.org/10.1074/jbc.271.37.22663
Capdevila, Jorge H. ; Wei, Shozou ; Helvig, Christian ; Falck, J R ; Belosludtsev, Yuri ; Truan, Gilles ; Graham-Lorence, Sandra E. ; Peterson, Julian A. / The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 37. pp. 22663-22671.
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abstract = "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 regioand 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 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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AU - Capdevila, Jorge H.

AU - Wei, Shozou

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AU - Belosludtsev, Yuri

AU - Truan, Gilles

AU - Graham-Lorence, Sandra E.

AU - Peterson, Julian A.

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N2 - 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 regioand 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 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).

AB - 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 regioand 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 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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