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• W2004201056 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 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 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). 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 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). Miura and Fulco (1Miura Y. Fulco A.J. J. Biol. Chem. 1974; 249: 1880-1888Google Scholar, 2Miura Y. Fulco A.J. Biochim. Biophys. Acta. 1975; 388: 305-317Google Scholar) originally reported by that extracts of Bacillus megaterium could monooxygenate fatty acids. The enzyme responsible for this reaction was isolated, purified, cloned, sequenced, and found to be a 120-kDa fusion protein of a class II cytochrome P450 and NADPH-P450 reductase and was named cytochrome P450BM-3 (P450BM-3) 1The abbreviations used are: P450BM-3, CYP102the soluble, bacterial P450 isolated from Bacillus megateriumP450BM-Pthe hemoprotein domain of P450BM-3EETcis-epoxyeicosatrienoic acidEPAeicosapentaenoic acidETAeicosatrienoic acid16-, 17-, 18-, 19-, and 20-OH-AA16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acid, respectivelyHPLChigh pressure liquid chromatographyRPreverse phaseNPnormal phasePTLCpreparative thin layer chromatographyGCgas chromatographyMSmass spectrometryNICInegative ion electron capture chemical ionizationMemethyl esterTMStrimethylsilyl etherPFBpentafluorobenzyl esterMOPS3-(N-morpholino)propanesulfonic acidRtretention time. (3Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Google Scholar, 4Wen L.-P. Fulco A.J. J. Biol. Chem. 1987; 262: 6676-6682Google Scholar). P450BM-3 contains heme, FAD, and FMN in a stoichiometry of 1:1:1, respectively (3Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Google Scholar). Both purified and recombinant P450BM-3 catalyze NADPH-dependent oxidation of medium and long chain saturated fatty acids, with optimum chain lengths of 14-16 carbons (3Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Google Scholar, 5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar). The regiochemistry of fatty acid hydroxylation by P450BM-3 is more or less chain length-dependent, i.e. as chain length increases, regioselectivity shifts from the Ω-1 to the Ω-2 carbon of the fatty acid (5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar). The turnover number of purified P450BM-3, in the monooxygenation of palmitic acid, is approximately 1.6 µmol/min/nmol at 25°C (3Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Google Scholar, 5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar), which is similar to that of P450cam for camphor hydroxylation under similar conditions (6Peterson J.A. Mock D.M. Cooper D.Y. Rosenthal O. Snyder R. Witmer C. Cytochromes P450 and b5. Plenum Press, New York1975: 311Google Scholar) and 100-1,000 times greater than that seen for most substrates with mammalian P450s. Monooxygenation of fatty acids by P450BM-3 is tightly coupled with a stoichiometry of NADPH, oxygen, and fatty acid consumed to hydroxylated fatty acid product formed of 1:1:1:1 (5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar). Under conditions of limiting substrate, P450BM-3 will further oxidize the initial metabolites to products that include diols and keto-alcohols (7Boddupalli S.S. Pramanik B.C. Slaughter C.A. Estabrook R.W. Peterson J.A. Arch. Biochem. Biophys. 1992; 292: 20-28Google Scholar). the soluble, bacterial P450 isolated from Bacillus megaterium the hemoprotein domain of P450BM-3 cis-epoxyeicosatrienoic acid eicosapentaenoic acid eicosatrienoic acid 16-, 17-, 18-, 19-, and 20-hydroxyeicosatetraenoic acid, respectively high pressure liquid chromatography reverse phase normal phase preparative thin layer chromatography gas chromatography mass spectrometry negative ion electron capture chemical ionization methyl ester trimethylsilyl ether pentafluorobenzyl ester 3-(N-morpholino)propanesulfonic acid retention time. In eukaryotes, AA serves both a structural role as a component of cellular membranes, and a critical functional role by participating in a variety of receptor/agonist-mediated signaling cascades (8Smith W.L. Am. J. Physiol. 1992; 263: F181-F191Google Scholar). The latter role is a consequence of regio- and stereoselective oxygenations of AA that is utilized by mammalian cells to transduce the signal (8Smith W.L. Am. J. Physiol. 1992; 263: F181-F191Google Scholar, 9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 10McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 339-369Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 12Harder D.R. Campbell W.B. Roman R.J. J. Vasc. Res. 1995; 32: 79-92Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar). The transduced chemical information is then decoded either by receptor mediated processes or, alternatively, by the direct effects of the oxygenated products on metabolic pathways (8Smith W.L. Am. J. Physiol. 1992; 263: F181-F191Google Scholar, 9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 10McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 339-369Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 12Harder D.R. Campbell W.B. Roman R.J. J. Vasc. Res. 1995; 32: 79-92Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar). In addition to prostaglandin synthases and lipoxygenases, well recognized members of the AA cascade, the contribution of microsomal P450 to the metabolism of endogenous AA pools is now well established (9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 10McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 339-369Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 12Harder D.R. Campbell W.B. Roman R.J. J. Vasc. Res. 1995; 32: 79-92Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar). Eukaryotic P450s oxidize AA by one or more types of reactions: 1) allylic oxidation to generate six different regioisomeric hydroxyeicosatetraenoic acids containing a cis,trans-conjugated dienol, 2) hydroxylation at sp3 carbons near or at the methyl terminus to generate 16-, 17-, 18-, 19-, or 20-OH-AA, and 3) olefinic bond epoxidation to generate four regioisomeric EETs. While the physiological significance of allylic oxidation remains obscure, products of hydroxylation and olefinic bond epoxidation reactions display a variety of potent biological activities and have been implicated in processes ranging from hormonal signaling to the pathophysiology of hypertension (9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 10McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31: 339-369Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 12Harder D.R. Campbell W.B. Roman R.J. J. Vasc. Res. 1995; 32: 79-92Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar). As with other metabolites of the AA cascade, e.g. prostanoids and leukotrienes, regio- and stereochemical features define biological activity and/or potency. Thus, understanding active site topology and the structural determinants of asymmetric catalysis by P450s are prerequisites for modifying their mechanism of action, and ultimately, for rational pharmacological intervention. Studies using microsomal fractions, and/or solubilized and purified mammalian P450 isoforms have demonstrated that the hemoprotein controls the regio- and stereoselectivities of oxidation of AA in an isoform-specific fashion at three different levels: 1) the type of reaction catalyzed, i.e. epoxidation to form EETs by CYP2B and CYP2C isoforms (9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar), or hydroxylation at C-16, C-17, and C-18 by CYP1A1 and CYP1A2 isoforms (9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar), and at C-19 and C-20 by the CYP4A isoform (11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar, 14Imaoka S. Tanaka S. Funae Y. Biochem. Int. 1989; 18: 731-740Google Scholar, 15Roman L.J. Palmer C.N. Clark J.E. Muerhoff A.S. Griffin K.J. Johnson E.F. Masters B.S. Arch. Biochem. Biophys. 1993; 307: 57-65Google Scholar); 2) positional selectivity, i.e. differentiation among four chemically equivalent olefinic bonds or between five sp3 hybridized carbons (hydroxylations at C-16-C-20) (11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar); and 3) absolute configuration. These processes are all the more remarkable since AA is an unbiased, acyclic molecule with high rotational freedom, and it is epoxidized by several mammalian P450 isoforms with unprecedented stereoselectivity (9Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6: 731-736Google Scholar, 11Oliw E.H. Prog. Lipid Res. 1994; 33: 329-354Google Scholar, 13Capdevila J.H. Zeldin D. Makita K. Karara A. Falck J.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. 2nd Ed. Plenum Press, New York1995: 443Google Scholar). As a guide to the protein structural determinants of polyunsaturated fatty acid metabolism by P450s, we describe herein the highly regio- and stereoselective metabolism of AA by recombinant P450BM-3, one of the four soluble P450s for which an atomic structure has been determined (16Cupp-Vickery J.R. Poulos T.L. Nat. Struct. Biol. 1995; 2: 144-153Google Scholar, 17Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Google Scholar, 18Hasemann C.A. Ravichandran K.G. Peterson J.A. Deisenhofer J. J. Mol. Biol. 1994; 236: 1169-1185Google Scholar, 19Ravichandran K.G. Boddupalli S.S. Hasemann C.A. Peterson J.A. Deisenhofer J. Science. 1993; 261: 731-736Google Scholar). Based on these results and similar analyses done with the AA cogeners EPA and ETA, we propose a model of the active site that accounts for the asymmetric catalysis by P450BM-3. The fatty acids were obtained from NuCheck Prep, Inc. All other chemicals were obtained in the purest form available from Sigma. The original plasmid, containing the gene encoding P450BM-3, was a gift from Dr. A. Fulco (Department of Biochemistry, UCLA, Los Angeles, CA). In preparation for our studies of the mechanism of P450BM-3, we constructed a new expression plasmid that contained only the gene encoding P450BM-3 and no extraneous B. megaterium DNA. We had previously constructed a plasmid for the expression of the heme domain of P450BM-3 and a construct from this project, pIBI-BMP2 (20Boddupalli S.S. Oster T. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1992; 267: 10375-10380Google Scholar), was linearized with SalI. Likewise, a vector had been constructed for the expression of the reductase domain of P450BM-3, pIBI-BMR (21Oster T. Boddupalli S.S. Peterson J.A. J. Biol. Chem. 1991; 266: 22718-22725Google Scholar). A 1.78-kilobase pair fragment from SalI-digested pIBI-BMR was recovered and ligated into the linearized pIBI-BMP2, giving pIBI-BM3. The sequence of the gene encoding P450BM-3 in this plasmid was confirmed. The plasmid was used to transform Escherichia coli strain DH5α. The protein was overexpressed and purified from these cells using published procedures (5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar). The concentration of the purified protein was estimated from the difference absorbance spectrum of the carbonyl complex of the ferrous form versus the ferrous form using the molar absorptivity of 91 mM-1cm-1 for the wavelength pair of 450 versus 490 nm (22Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Google Scholar). The substrate binding spectra used for the determination of spectral binding constants (Ks), were recorded with a Hewlett Packard diode array spectrophotometer maintained at 25°C. The absorbance data for substrate binding were analyzed with the program Microsoft Excel.© The rates of P450BM-3-dependent oxygen and NADPH utilization were measured using 20-50 nM solutions of the enzyme in 50 mM MOPS buffer, pH 7.4, containing 100 µM fatty acid. After a 5-min preincubation, reactions were started by addition of NADPH. Oxygen concentration was measured with a Clark-type oxygen electrode instrument (Yellow Springs Instrument Co.). NADPH concentration was measured spectrophotometrically at 340 nm (ϵ = 6.22 mM-1 cm-1). For product quantification and structural characterization, incubations were performed at 30°C under atmospheric air and with vigorous mixing. Reaction mixtures in 50 mM Tris-Cl buffer, pH 7.4, containing 10 mM MgCl2, 150 mM KCl, 8 mM sodium isocitrate, isocitrate dehydrogenase (1.0 IU/ml), dilauroyl phosphatidyl choline (0.05 µg/ml), and P450BM-3 (2-10 nM, final concentration) were incubated 2.5 min prior to the addition of the sodium salts of either AA, EPA, or ETA (25 mM each in 0.05 mM Tris-Cl buffer, pH 8.0) to final concentrations of 50-100 µM each. After 1 min, reactions were started by the addition of NADPH (1 mM, final concentration). At different time points, aliquots were withdrawn, and the organic soluble products were extracted three times with equal volumes of ethyl ether containing HOAc (0.05%, v/v). After solvent evaporation under a stream of nitrogen, the products were resolved by RP-HPLC on a 5-µm Dynamax Microsorb C18 column (4.6 × 250 mm, Rainin Instruments Co., Woburn, MA) using a linear solvent gradient from 49.9% CH3CN, 49.9% H2O, 0.1% HOAc to 99.9% CH3CN, 0.1% HOAc over 40 min at 1 ml/min. Products were quantified by on-line liquid scintillation using a Radiomatic Flo-One β-Detector (Radiomatic Instruments, Tampa, FL). The identification of 18-OH-AA and of 14,15-EET was done using published methodology (23Capdevila J.H. Falck J.R. Dishman E. Karara A. Methods Enzymol. 1990; 187: 385-394Google Scholar, 24Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Google Scholar, 25Falck J.R. Lumin S. Blair I. Dishman E. Martin M.V. Waxman D.J. Guengerich F.P. Capdevila J.H. J. Biol. Chem. 1990; 265: 10244-10249Google Scholar, 26Kuhn H. Schewe T. Rapoport S.M. Brash A.R. Basic Life Sci. 1988; 49: 945-949Google Scholar) and confirmed with synthetic standards. Synthetic 16-, 17-, and 18-OH-AAs were resolved by normal phase HPLC on a 5-µm Dynamax Microsorb Silica column (4.6 × 250 mm, Rainin Instruments) using an isocratic solvent mixture composed of 0.4% 2-propanol, 0.1% HOAc, 99.5% hexane at 2 ml/min (Rt ~ 31.5, 33.1, and 36.5 min for 17-, 18-, and 16-OH-AA, respectively). Synthetic 19- and 20-OH-AAs were resolved by NP-HPLC as above using a solvent mixture of 1% 2-propanol, 0.1% HOAc, 98.9% hexane at 3 ml/min (Rt ~ 14.6 and 20.8 min for 19- and 20-OH-AA, respectively). The enantiomers of methyl 14,15-epoxyeicosatrienoate were resolved by chiral phase HPLC as described previously (24Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Google Scholar). For the characterization of EPA metabolites, the organic soluble material extracted from solutions containing [1-14C]EPA (100 µM final concentration, 0.1 µCi/µmol), 5 nM P450BM-3, and 1 mM NADPH was resolved by RP-HPLC as described. The radioactive fraction eluting from the HPLC column with the retention time of authentic 17,18-epoxy-EPA (18.7 min) was collected batchwise and further characterized. To confirm the epoxide nature of the metabolite, an aliquot of the purified material (2-5 µg) was incubated, under an argon atmosphere and with constant mixing, with 0.25 ml of a mixture containing 20% EtOH, 40% H2O, and 40% glacial HOAc. After 12 h at room temperature, the reaction mixture was diluted with 1 ml of 0.1 M KCl and extracted twice with equal volumes of ethyl ether. The resulting product co-eluted in RP-HPLC with synthetic vic-17,18-dihydroxy-5, 8,11,14-eicosatetraenoic acid (Rt ≈ 10 min) and, after derivatization to the corresponding TMS ether, PFB ester, showed a NICI/GC/MS fragmentation pattern identical to that of an authentic standard (Fig. 1A). For regiochemical analysis, an aliquot of the hydrated epoxide (5 µg) was hydrogenated over PtO2, derivatized to the corresponding PFB ester (24Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Google Scholar), and purified by SiO2 chromatography. The dry residue was dissolved in 200 µl of NaIO4 (10 mg/ml in 70% CH3OH) and, after 2 h at 50°C, the product was extracted into hexane and purified by RP-HPLC using a linear solvent gradient from 49.9% CH3CN, 49.9% H2O, 0.1% HOAc, to 99.9% CH3CN, 0.1% HOAc over 40 min at 1 ml/min (Rt~ 43 min). The purified aldehyde, resulting from oxidative cleavage of the vic-diol precursor, was dried under a stream of N2, mixed with 200 µl of 0.5% solution of methoxylamine hydrochloride in pyridine (Pierce), incubated 3 h at 30°C, extracted into hexane, and then characterized by NICI/GC/MS (Fig. 1B). For structural analysis, the organic soluble products extracted from solutions containing [1-14C]ETA (100 µM, final concentration, 0.2 µCi/µmol), 2 nM P450BM-3, and 1 mM NADPH were purified by RP-HPLC as above. The radioactive material eluting from the RP-HPLC column between 18 and 20 min was collected batchwise and, after solvent evaporation, resolved into fractions a, b, and c (Rt ~ 16.9, 23.1, and 37.1 min, respectively for a, b, and c) by NP-HPLC on a 5-µm Dynamax Microsorb Silica column (4.6 × 250 mm) using an isocratic solvent mixture composed of 0.5% 2-propanol, 0.1% HOAc, 99.4% hexane at a flow rate of 2 ml/min. After catalytic hydrogenation and derivatization to the corresponding PFB esters, fractions a, b, and c were characterized by NP-HPLC on the above silica column using an isocratic solvent mixture composed of 0.2% 2-propanol, 99.8% hexane at a flow rate of 2 ml/min. In this chromatographic system, the PFB esters of hydrogenated a, b, and c (Rt ~ 20, 23, and 35 min, respectively) co-eluted with the PFB esters of synthetic 17-, 18-, and 19-hydroxyeicosanoic acid, respectively. The PFB esters of hydrogenated a, b, and c were further characterized by NICI/GC/MS. To a room temperature mixture of synthetic methyl 18-hydroxyeicosatetraenoate (1.5 mg) (27Falck J.R. Lumin S. Lee S.-G. Heckmann B. Mioskowski C. Karara A. Capdevila J.H. Tetrahedron Lett. 1992; 33: 4893-4896Google Scholar), R-(+)-α-methoxy-α-trifluoromethylphenylacetic acid (1.5 mg), and dimethylaminopyridine (0.2 mg) in anhydrous CH2Cl2 (1 ml) was added 1,3-dicyclohexylcarbodiimide (1.5 mg) in one portion with stirring. After 12 h, the solvent was removed in vacuo, and the residue was purified by PTLC (SiO2: 20% EtOAc/hexane, RF ≈ 0.59). A sample of enzymatically derived 18-hydroxyeicosatetraenoic acid (0.8 mg) was esterified with excess diazomethane in Et2O for 1 h prior to derivatization exactly as described above. The individual Mosher esters were further purified by HPLC using a Dynamax Microsorb Silica column (4.6 × 250 mm) isocratically eluted with 0.35% EtOH, 99.65% hexane at 4 ml/min with UV monitoring at 210 nm. Comparative analysis by co-injection on a Chiracel OD HPLC column (4.6 × 250 mm, J. T. Baker Inc.) isocratically eluted with 0.2% 2-propanol, 99.8% hexane at 2 ml/min with UV monitoring showed the 18(S)-isomer eluted with a retention time of 6.6 min, whereas the 18(R)-isomer and the enzymatically derived product co-eluted with a retention time of 5.8 min. A stream of O3 in oxygen was passed for 2 h through a solution of enzymatically generated 17,18-epoxyeicosatetraenoic acid (1.87 mg) in 90% CH3OH, 10% CH2Cl2 cooled to 0°C. NaBH4 (2 mg) was added, and the mixture was stirred at room temperature. After 30 min, the reaction mixture was diluted with Et2O (2 ml) and H2O (2 ml). The organic phase was separated, and the aqueous phase was extracted once more with Et2O. The combined organic phases were evaporated in vacuo, and the residue was dried azeotropically with anhydrous benzene, then dissolved in dry pyridine (100 µl) to which was added benzoyl chloride (15 µl). After 12 h, the reaction mixture was diluted with CH2Cl2 (2 ml), and a saturated aqueous solution of CuSO4 (300 µl) was added to effect phase separation. The organic layer was collected and concentrated in vacuo, and the residue was purified by PTLC (15% EtOAc, 85% hexane, RF ~ 0.26) using standards of synthetic epoxy-benzoate in adjacent lanes as guides to the location of the enzymatically derived 3,4-epoxyhexan-1-yl benzoate. A chiral standard of the 3,4-epoxyhexan-1-yl benzoate was prepared by Sharpless asymmetric epoxidation of (3Z)-hexen-1-ol as described previously (28Rossiter B.E. Sharpless K.B. J. Org. Chem. 1984; 49: 3707-3711Google Scholar) affording (3R,4S)-epoxyhexan-1-ol, [α]22D+7.27° (c 1.85, CHCl3), as a colorless oil in 45% yield. The epoxy-alcohol (58 mg, 0.5 mmol) was dissolved in dry pyridine (1 ml) and cooled to 0°C, and benzoyl chloride (58 µl, 0.75 mmol, 1.5 equivalent) was added. After stirring at room temperature for 12 h, the reaction mixture was diluted with CH2Cl2 (10 ml), washed with saturated aqueous CuSO4 solution (3 × 5 ml), brine (5 ml), dried over Na2SO4, and evaporated in vacuo. The residue was purified by PTLC (SiO2: 15% EtOAc, 85% hexane, RF ≈ 0.26) to give the epoxy-benzoate (98 mg, 100%) as a colorless oil. 1H NMR (CDCl3, 250 MHz): δ 1.06 (t, 3H, 6.6 Hz), 1.49-1.69 (m, 2H), 1.90-2.14 (m, 2H), 2.90-2.98 (m, 1H), 3.09-3.18 (m, 1H), 4.53 (t, 2H, 6.6 Hz), 7.46 (t, 2H, 7.7 Hz), 7.56 (apparent t, 1H, 7.7 Hz), 8.07 (d, 2H, 7.7 Hz). The (3S,4R)-enantiomer was obtained analogously. Comparisons using a Chiracel OC HPLC column (4.6 × 250 mm) eluted isocratically with 0.2% 2-PrOH, 0.1% EtOH, 99.7% hexane at 1.1 ml/min with UV monitoring at 210 nm showed the (3R,4S)-isomer had a retention time of 58 min, whereas the (3S,4R)-enantiomer and the biologically derived sample co-eluted with a retention time of 45 min. Samples were dissolved in dodecane and analyzed by NICI/GC/MS on a Nermag R1010C quadrupole instrument interfaced to a Varian Vista Gas chromatograph utilizing He and CH4 as carrier and reagent gases, respectively. Splitless injections were made onto a 30-m SPB-1 fused silica capillary column (0.32-mm inner diameter, 0.25-µm coating thickness, Supelco Inc. Bellefonte, PA). After 1 min at 100°C, the oven temperature was raised to 300°C at 10°C/min. The rate of fatty acid oxidation by P450BM-3 can be easily measured either polarographically using an oxygen electrode or spectrophotometrically monitoring absorbance changes at 340 nm. Seen in Fig. 2 is an oxygen electrode trace of O2 consumption during the NADPH-dependent metabolism of AA by P450BM-3. The order of addition of reactants is crucial, i.e. NADPH must be added last to avoid inactivating the reductase domain of P450BM-3 (3Narhi L.O. Fulco A.J. J. Biol. Chem. 1986; 261: 7160-7169Google Scholar, 29Sevrioukova I.F. Peterson J.A. Biochimie (Paris). 1995; 77: 562-572Google Scholar). During the first few seconds after addition of NADP, the turnover number of P450BM-3 is 3.5 µmol of O2 consumed/min/nmol, but decreases as oxidized products accumulate (Fig. 2). In experiments not shown here, the rate of NADPH oxidation, measured at 340 nm, was approximately the same as the rate of oxygen consumption per mol of P450 per min indicating a tight coupling of NADPH oxidation, O2 consumption and substrate oxidation. Within the first 30 s of incubation, the rates of AA utilization and product formation began to decrease showing the lack of a clear linear relationship between product formation and incubation time. As the rate of AA oxidation decreased, the recovery of polyoxygenated products, derived from secondary oxygenations, increased concomitantly, and became predominant 2-3 min after initiation. When limiting amounts of AA were added, as shown in Fig. 2, the ratio of O2 consumed per mol of AA added was approximately 2, indicating that fatty acid polyoxygenation had occurred as shown for palmitic acid (5Boddupalli S.S. Estabrook R.W. Peterson J.A. J. Biol. Chem. 1990; 265: 4233-4239Google Scholar, 7Boddupalli S.S. Pramanik B.C. Slaughter C.A. Estabrook R.W. Peterson J.A. Arch. Biochem. Biophys. 1992; 292: 20-28Google Scholar). The limited solubility of AA made impractical attempts to increase its concentration, and thus prolong enzyme-substrate saturation. The rates of product formation shown in Table I were obtained at 30°C and after a 30 s incubation, and are approximations of the initial velocities. They are useful only for comparative purposes.TABLE I.Reaction rates and spectral binding constants of P450BM-3 using various substratesSubstrateReaction rateaReaction rates in µmols of product formed/min/nmol of P450BM-3.Spectral binding constant, KsµMAA3.2 ± 0.41.2 ± 0.1EPA1.4 ± 0.21.6 ± 0.5ETA2.9 ± 0.1NDbNot determined.a Reaction rates in µmols of product formed/min/nmol of P450BM-3.b Not determined. Open table in a new tab Among the fatty acids metabolized by P450BM-3, AA showed the highest oxidation rate, 3.2 µmol of product formed/min/nmol of P450BM-3 (Table I). To the best of our knowledge, the rates at which P450BM-3 catalyzes the redox coupled activation of molecular oxygen, the cleavage of the oxygen-oxygen bond, and the insertion of a reacti" @default.
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