Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase
2007; Elsevier BV; Volume: 49; Issue: 2 Linguagem: Inglês
10.1194/jlr.m700514-jlr200
ISSN1539-7262
Autores Tópico(s)Biochemical and biochemical processes
ResumoManganese lipoxygenase (Mn-LOX) catalyzes the rearrangement of bis-allylic S-hydroperoxides to allylic R-hydroperoxides, but little is known about the reaction mechanism. 1-Linoleoyl-lysoglycerophosphatidylcholine was oxidized in analogy with 18:2n-6 at the bis-allylic carbon with rearrangement to C-13 at the end of lipoxygenation, suggesting a "tail-first" model. The rearrangement of bis-allylic hydroperoxides was influenced by double bond configuration and the chain length of fatty acids. The Gly316Ala mutant changed the position of lipoxygenation toward the carboxyl group of 20:2n-6 and 20:3n-3 and prevented the bis-allylic hydroperoxide of 20:3n-3 but not 20:2n-6 to interact with the catalytic metal. The oxidized form, MnIII-LOX, likely accepts an electron from the bis-allylic hydroperoxide anion with the formation of the peroxyl radical, but rearrangement of 11-hydroperoxyoctadecatrienoic acid by Mn-LOX was not reduced in D2O (pD 7.5), and aqueous Fe3+ did not transfer 11S-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid to allylic hydroperoxides. Mutants in the vicinity of the catalytic metal, Asn466Leu and Ser469Ala, had little influence on bis-allylic hydroperoxide rearrangement. In conclusion, Mn-LOX transforms bis-allylic hydroperoxides to allylic by a reaction likely based on the positioning of the hydroperoxide close to Mn3+ and electron transfer to the metal, with the formation of a bis-allylic peroxyl radical, β-fragmentation, and oxygenation under steric control by the protein. Manganese lipoxygenase (Mn-LOX) catalyzes the rearrangement of bis-allylic S-hydroperoxides to allylic R-hydroperoxides, but little is known about the reaction mechanism. 1-Linoleoyl-lysoglycerophosphatidylcholine was oxidized in analogy with 18:2n-6 at the bis-allylic carbon with rearrangement to C-13 at the end of lipoxygenation, suggesting a "tail-first" model. The rearrangement of bis-allylic hydroperoxides was influenced by double bond configuration and the chain length of fatty acids. The Gly316Ala mutant changed the position of lipoxygenation toward the carboxyl group of 20:2n-6 and 20:3n-3 and prevented the bis-allylic hydroperoxide of 20:3n-3 but not 20:2n-6 to interact with the catalytic metal. The oxidized form, MnIII-LOX, likely accepts an electron from the bis-allylic hydroperoxide anion with the formation of the peroxyl radical, but rearrangement of 11-hydroperoxyoctadecatrienoic acid by Mn-LOX was not reduced in D2O (pD 7.5), and aqueous Fe3+ did not transfer 11S-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid to allylic hydroperoxides. Mutants in the vicinity of the catalytic metal, Asn466Leu and Ser469Ala, had little influence on bis-allylic hydroperoxide rearrangement. In conclusion, Mn-LOX transforms bis-allylic hydroperoxides to allylic by a reaction likely based on the positioning of the hydroperoxide close to Mn3+ and electron transfer to the metal, with the formation of a bis-allylic peroxyl radical, β-fragmentation, and oxygenation under steric control by the protein. Lipoxygenases constitute a gene family of nonheme metalloenzymes with conserved metal ligands, which oxygenate polyunsaturated fatty acids to hydroperoxides and related compounds (1Brash A.R. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate.J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1131) Google Scholar). Lipoxygenases occur in plants and mammals and are of considerable medical and biological interest (2Kuhn H. Saam J. Eibach S. Holzhütter H.G. Ivanov I. Walther M. Structural biology of mammalian lipoxygenases: enzymatic consequences of targeted alterations of the protein structure.Biochem. Biophys. Res. Commun. 2005; 338: 93-101Crossref PubMed Scopus (95) Google Scholar, 3Liavonchanka A. Feussner I. Lipoxygenases: occurrence, functions and catalysis.J. Plant Physiol. 2006; 163: 348-357Crossref PubMed Scopus (334) Google Scholar). Lipoxygenation occurs by hydrogen atom abstraction at the bis-allylic carbon of 1Z,4Z-pentadienes of fatty acids followed by O2 insertion, with the formation of cis-trans conjugated (allylic) hydroperoxy fatty acids. Lipoxygenases occur in resting (reduced; Fe2+) and active (oxidized; Fe3+) forms. The active form of lipoxygenases contains the catalytic base, Fe3+OH− (4Tomchick D.R. Phan P. Cymborowski M. Minor W. Holman T.R. Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.Biochemistry. 2001; 40: 7509-7517Crossref PubMed Scopus (127) Google Scholar), which forms a carbon-centered radical (L·). The latter reacts with molecular oxygen and forms a peroxyl radical: Fe3+OH-+LH→Fe2+OH2+L.(Eq. 1) L.+O2⇆LOO.(Eq. 2) In the next step, the catalytic base is regenerated, with the formation of the hydroperoxide: LOO.+Fe2+OH2→Fe3+OH+LOOH(Eq. 3) Experimental and density functional studies of H atom abstraction by soybean lipoxygenase (sLO-1) suggest a proton-coupled electron transfer mechanism by which the electron and the proton are transferred separately (5Lehnert N. Solomon E.I. Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase.J. Biol. Inorg. Chem. 2003; 8: 294-305Crossref PubMed Scopus (117) Google Scholar, 6Hammes-Schiffer S. Hydrogen tunneling and protein motion in enzyme reactions.Acc. Chem. Res. 2006; 39: 93-100Crossref PubMed Scopus (199) Google Scholar, 7Hatcher E. Soudackov A.V. Hammes-Schiffer S. Proton-coupled electron transfer in soybean lipoxygenase: dynamical behavior and temperature dependence of kinetic isotope effects.J. Am. Chem. Soc. 2007; 129: 187-196Crossref PubMed Scopus (137) Google Scholar).Autoxidation of polyunsaturated fatty acids also occurs by the abstraction of hydrogen at bis-allylic carbons, with the formation of peroxyl radicals, yet lipoxygenation and autoxidation differ in other respects. Lipoxygenases form cis-trans conjugated hydroperoxides with stereo and position selectivity, and the initial hydrogen abstraction is associated with a large kinetic isotope effect (kH/kD ∼ 40) that is attributable to tunneling of the deuterium atom (8Rickert K.W. Klinman J.P. Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects.Biochemistry. 1999; 38: 12218-12228Crossref PubMed Scopus (179) Google Scholar). The corresponding kinetic isotope effect during autoxidation is in agreement with classical theory (kH/kD 5–6) (9Kitaguchi H. Ohkubo K. Ogo S. Fukuzumi S. Additivity rule holds in the hydrogen transfer reactivity of unsaturated fatty acids with a peroxyl radical: mechanistic insight into lipoxygenase.Chem. Commun. (Camb.). 2006; : 979-981Crossref PubMed Scopus (15) Google Scholar). Autoxidation of polyunsaturated fatty acids generates both unconjugated and cis-trans and trans-trans conjugated hydroperoxides (10Chan D.W-S. Levett G. Matthew J.A. The mechanism of the rearrangement of linoleate hydroperoxides.Chem. Phys. Lipids. 1979; 24: 245-256Crossref Scopus (115) Google Scholar, 11Brash A.R. Autoxidation of methyl linoleate: identification of the bis-allylic 11-hydroperoxide.Lipids. 2000; 35: 947-952Crossref PubMed Scopus (63) Google Scholar, 12Pratt D.A. Mills J.H. Porter N.A. Theoretical calculations of carbon-oxygen bond dissociation enthalpies of peroxyl radicals formed in the autoxidation of lipids.J. Am. Chem. Soc. 2003; 125: 5801-5810Crossref PubMed Scopus (153) Google Scholar). The spin density of the carbon-centered radical is highest at the bis-allylic carbons (13Tallman K.A. Pratt D.A. Porter N.A. Kinetic products of linoleate peroxidation: rapid beta-fragmentation of nonconjugated peroxyls.J. Am. Chem. Soc. 2001; 123: 11827-11828Crossref PubMed Scopus (89) Google Scholar) and the major products are formed by oxygenation at these positions. Rearrangement of these bis-allylic peroxyl radicals to allylic radicals occurs rapidly, with a rate constant of ∼2 × 106, ∼105 times faster than the rearrangement of alkyl radicals. Consequently, bis-allylic hydroperoxides accumulate only during autoxidation in the presence of high concentrations of hydrogen donors (e.g., α-tocopherol), which convert the bis-allylic peroxyl radicals to the relatively stable bis-allylic hydroperoxides (11Brash A.R. Autoxidation of methyl linoleate: identification of the bis-allylic 11-hydroperoxide.Lipids. 2000; 35: 947-952Crossref PubMed Scopus (63) Google Scholar).Manganese lipoxygenase (Mn-LOX) is secreted by the take-all fungus and contains Mn as the catalytic metal (14Su C. Oliw E.H. Manganese lipoxygenase. Purification and characterization.J. Biol. Chem. 1998; 273: 13072-13079Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). It is the only known naturally occurring lipoxygenase that forms significant amounts (>0.5%) of bis-allylic hydroperoxides (15Hamberg M. Su C. Oliw E. Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.J. Biol. Chem. 1998; 273: 13080-13088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Mn-LOX catalyzes the oxygenation of 18:2n-6 by suprafacial hydrogen abstraction at C-11 and O2 insertion at the bis-allylic position C-11 and, with double bond migration, at the allylic position C-13. The enzyme produces 11S-hydroperoxy-9Z,12Z-octadecadienoic acid (11-HPODE) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPODE) at an ∼1:4 ratio (15Hamberg M. Su C. Oliw E. Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.J. Biol. Chem. 1998; 273: 13080-13088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). At the end of lipoxygenation, 11-HPODE is transformed to 13-HPODE by Mn-LOX (15Hamberg M. Su C. Oliw E. Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.J. Biol. Chem. 1998; 273: 13080-13088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Electron paramagnetic resonance analysis suggests that the catalytic metal redox cycles (Mn2+/Mn3+) between the resting and active forms of the enzyme, in analogy with Fe-lipoxygenases, and that the metal coordinating residues are virtually identical (16Su C. Sahlin M. Oliw E.H. Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center.J. Biol. Chem. 2000; 275: 18830-18835Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 17Gaffney B.J. Su C. Oliw E.H. Assignment of EPR transitions in a manganese-containing lipoxygenase and prediction of local structure.Appl. Magn. Reson. 2001; 21: 413-424Crossref PubMed Scopus (20) Google Scholar, 18Hörnsten L. Su C. Osbourn A.E. Hellman U. Oliw E.H. Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.Eur. J. Biochem. 2002; 269: 2690-2697Crossref PubMed Scopus (38) Google Scholar, 19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar). To date, the oxygenation of bis-allylic carbons by Fe-lipoxygenases has only been reported for the recombinant lipoxygenase domain of allene oxide synthase of Plexaura homomalla, which transforms 20:3n-6 to the bis-allylic hydroperoxide at C-10 (∼5%) and to the allylic hydroperoxide at C-8 (20Boutaud O. Brash A.R. Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla.J. Biol. Chem. 1999; 274: 33764-33770Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Whether the bis-allylic hydroperoxide at C-10 was subject to rearrangement was not reported. 11-HPODE is a poor substrate of sLO-1 (15Hamberg M. Su C. Oliw E. Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.J. Biol. Chem. 1998; 273: 13080-13088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), but huge amounts of sLO-1 (36,600 U/ml, ∼0.25 mg/ml) catalyze the slow rearrangement of 11-HPODE (21Oliw E.H. Cristea M. Hamberg M. Biosynthesis and isomerization of 11-hydroperoxylinoleates by manganese- and iron-dependent lipoxygenases.Lipids. 2004; 39: 319-323Crossref PubMed Scopus (18) Google Scholar).It is of interest to compare the rearrangement of bis-allylic hydroperoxides to cis-trans conjugated hydroperoxides during autoxidation and Mn-LOX catalysis, as the reaction mechanisms may differ. A key intermediate is the unstable bis-allylic peroxyl radical, which transforms to an allylic peroxyl radical via β-fragmentation and oxygenation at an allylic position during autoxidation (11Brash A.R. Autoxidation of methyl linoleate: identification of the bis-allylic 11-hydroperoxide.Lipids. 2000; 35: 947-952Crossref PubMed Scopus (63) Google Scholar, 12Pratt D.A. Mills J.H. Porter N.A. Theoretical calculations of carbon-oxygen bond dissociation enthalpies of peroxyl radicals formed in the autoxidation of lipids.J. Am. Chem. Soc. 2003; 125: 5801-5810Crossref PubMed Scopus (153) Google Scholar): Lbis-allylicOO.⇆L.+O2(Eq. 4) L.+O2→LallylicOO.(Eq. 5) The LOO–H bond dissociation enthalpy is ∼88 kcal/mol, and it is likely that LOO· is formed during autoxidation by hydrogen abstraction by radical species (12Pratt D.A. Mills J.H. Porter N.A. Theoretical calculations of carbon-oxygen bond dissociation enthalpies of peroxyl radicals formed in the autoxidation of lipids.J. Am. Chem. Soc. 2003; 125: 5801-5810Crossref PubMed Scopus (153) Google Scholar). Mn-LOX catalyzes the rearrangement of bis-allylic hydroperoxides, likely by the formation of the bis-allylic peroxyl radical, β-fragmentation, and oxygen insertion at the n-6 (or n-8) position under steric control by the enzyme.How can bis-allylic peroxyl radicals be generated from bis-allylic hydroperoxides by Mn-LOX? The mechanism is likely related to the high redox potential of Mn3+. It has been suggested that peroxyl radicals might be generated from peroxide anions by the reduction of metal ions (22Davies M.J. Slater T.F. Studies on the metal-ion and lipoxygenase-catalysed breakdown of hydroperoxides using electron-spin-resonance spectroscopy.Biochem. J. 1987; 245: 167-173Crossref PubMed Scopus (76) Google Scholar), suggesting the following hypothetical rearrangement mechanism for Mn-LOX: Mn3+OH-+Lbis-allylicOO-+H+→Mn2+OH2+Lbis-allylicOO.(Eq. 6) The bis-allylic peroxyl radical is then subjected to β-fragmentation and oxygen insertion under steric control by the enzyme, with formation of an allylic peroxyl radical as discussed above. Recently, hypochloric acid (1 mM) was reported to convert allylic hydroperoxides of 18:2n-6 to peroxyl radicals, but the mechanism is unknown (23Miyamoto S. Martinez G.R. Rettori D. Augusto O. Medeiros M.H. Di Mascio P. Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl radical intermediates and singlet molecular oxygen.Proc. Natl. Acad. Sci. USA. 2006; 103: 293-298Crossref PubMed Scopus (108) Google Scholar). In contrast, homolytic cleavage of hydroperoxides has been studied extensively. Aqueous Fe2+, other divalent metal ions, hematin, and the reduced forms of Fe-lipoxygenases and Mn-LOX can catalyze the homolytic cleavage of alkyl hydroperoxides (24Yu Z. Schneider C. Boeglin W.E. Marnett L.J. Brash A.R. The lipoxygenase gene ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase.Proc. Natl. Acad. Sci. USA. 2003; 100: 9162-9167Crossref PubMed Scopus (117) Google Scholar, 25Cristea M. Oliw E.H. A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols.J. Biol. Chem. 2006; 281: 17612-17623Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Homolytic cleavage by Mn-LOX is influenced by the position of the hydroperoxide at the active site, as judged from the effects of substrate chain length and double bond configuration (25Cristea M. Oliw E.H. A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols.J. Biol. Chem. 2006; 281: 17612-17623Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). It seemed possible that these factors also might influence the rearrangement of bis-allylic hydroperoxides to allylic.The aim of the present study was to study the factors controlling the rearrangement of bis-allylic hydroperoxides by Mn-LOX. The first goal was to confirm that the fatty acids bind "tail-first" in the active site of Mn-LOX during rearrangement in the same way as during oxygenation (26Cristea M. Oliw E.H. On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant.J. Lipid Res. 2007; 48: 890-903Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The second goal was to determine the influence of the Gly316Ala mutant on the rearrangement reaction. This mutant changed the position of oxygenation and the interaction of the hydroperoxides with the catalytic metal (25Cristea M. Oliw E.H. A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols.J. Biol. Chem. 2006; 281: 17612-17623Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). D2O could be expected to increase the Pka for peroxide anions by 0.4, and this solvent deuterium isotope effect might conceivably reduce both the concentration of the anion and the rate of rearrangement. Finally, we investigated whether mutants of presumed amino acids of the first and second coordinating spheres in a hydrogen bond network might affect the rearrangement. Asn694 and Glu697 of sLO-1 belong to this group of amino acids (4Tomchick D.R. Phan P. Cymborowski M. Minor W. Holman T.R. Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.Biochemistry. 2001; 40: 7509-7517Crossref PubMed Scopus (127) Google Scholar), and the homologous residues of Mn-LOX are Asn466 and Ser469 (18Hörnsten L. Su C. Osbourn A.E. Hellman U. Oliw E.H. Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.Eur. J. Biochem. 2002; 269: 2690-2697Crossref PubMed Scopus (38) Google Scholar, 19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar).EXPERIMENTAL PROCEDURESMaterials1-Palmitoyl-2-linoleoyl-glycerophosphatidylcholine (GPC; 99%), Lα-lysoGPC (from soybean; 99%), dilinoleoyl-GPC (99%), 20:2n-6 (99%), 20:3n-3 (99%), and 20:4n-6 (99%) were obtained from Larodan (Malmö, Sweden). 18:2n-6 (99%), 18:3n-6 (99%), and HPLC solvents (Lichrosolve) were from Merck. Fatty acids were dissolved in ethanol and stored in stock solutions (50–100 mM) at −20°C; fresh solutions (50–150 μM) of the fatty acids were usually prepared in 0.1 M NaBO3 buffer (pH 9.0). Phospholipase A2 (bee venom), cholesteryl esterase (porcine pancreas), and 2H2O (99.98%) were from Sigma-Aldrich. Site-directed mutagenesis of Mn-LOX was performed as described, and the expression constructs were sequenced (19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar). Amino acids of Mn-LOX were numbered after cleavage of the N-terminal secretion signal of 16 amino acids from the Mn-LOX precursor of 618 amino acids (GenBank AAK81862) and the secreted enzyme of 602 amino acids was expressed (19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar). Recombinant Mn-LOX, Mn-LOX G316A, Mn-LOX N466L, and Mn-LOX S469A were expressed in Pichia pastoris (strain X-33) as secreted proteins using the expression vector pPICZAα (19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar). The recombinant enzymes were purified from the growth medium by hydrophobic interaction chromatography as described (14Su C. Oliw E.H. Manganese lipoxygenase. Purification and characterization.J. Biol. Chem. 1998; 273: 13072-13079Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar), concentrated by diafiltration, and stored at +4°C.Mn-LOX assayLight absorbance was measured with a dual-beam spectrophotometer (Shimadzu UV-2101PC) with a 1 cm path length. The cis-trans conjugated hydro(pero)xy fatty acids were assumed to have an extinction coefficient of 25,000 cm−1 M−1 (27Graff G. Anderson L.A. Jaques L.W. Preparation and purification of soybean lipoxygenase-derived unsaturated hydroperoxy and hydroxy fatty acids and determination of molar absorptivities of hydroxy fatty acids.Anal. Biochem. 1990; 188: 38-47Crossref PubMed Scopus (97) Google Scholar). Lipoxygenase activity was monitored by ultraviolet (UV) spectroscopy (235–237 nm) in 0.1 M NaBO3 buffer (pH 9.0) with 50–120 μM substrate. Reaction was started by the addition of Mn-LOX. Oxygenated fatty acids were usually analyzed after extractive isolation (SepPak/C18; Waters) without acidification (19Cristea M. Engström Å. Su C. Hörnsten L. Oliw E.H. Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands.Arch. Biochem. Biophys. 2005; 434: 201-211Crossref PubMed Scopus (47) Google Scholar) and oxygenated 1-linoleoyl-lysoGPC after Folch extraction. The fatty acid hydroperoxides and keto fatty acids were reduced to alcohols by treatment with NaBH4 or NaB2H4 before LC-MS/MS analysis, unless stated otherwise. Mn-LOX was prepared in D2O buffer by repeated diafiltration (30 k; Amicon Ultra; Millipore) and added (in <1% volume) to 65 μM 11-hydroperoxyoctadecatrienoic acid (11-HPOTrE) in 0.1 M NaHPO4 (pD 7.5 and pH 7.5). pH was measured with glass electrodes from Radiometer Copenhagen. Because of the solvent isotope effect of deuterium oxide on pH glass electrodes, pD was calculated as measured pH plus 0.4 (28Salomaa P. Schaleger L.L. Long F.A. Solvent deuterium isotope effects on acid-base equilibria.J. Am. Chem. Soc. 1964; 86: 1-7Crossref Scopus (286) Google Scholar).HPLC-MS/MSReverse phase (RP)-HPLC with MS/MS analysis was performed with a Surveyor MS pump (Thermo) and with an octadecyl silica column (5 μm; 2.1 × 150 mm; Phenomenex or Hypersil Gold), which was usually eluted with methanol-water-acetic acid (750:250:0.06; Suprapur; Merck) at 0.3 ml/min. The effluent passed a photodiode array detector (Surveyor PDA; 5 cm path length) and was subjected to electrospray ionization in an ion trap mass spectrometer (LTQ; Thermo). The heated transfer capillary was set at 315°C, the ion isolation width at 1.5 amu, and the collision energy at 25–35 (arbitrary scale). Prostaglandin F1α (100 ng/min) was infused for tuning. 1-Linoleoyl-lysoGPC and its metabolites were separated on the Hypersil Gold column, which was eluted with acetonitrile-5 mM ammonium acetate (35:65) at 0.3 ml/min, with UV detection and MS/MS monitoring of positive ions.Normal phase (NP)-HPLC with MS/MS analysis was performed on silica with an analytical column (Kromasil-100SI; 250 × 2 mm, 5 μm, 100 Å), which was eluted at 0.3 ml/min with 5% isopropanol in hexane with 0.05 ml/l acetic acid (Constrametric 3200 pump; LDC). The effluent was combined with isopropanol-water (3:2; 0.2 ml/min) from a second pump (Surveyor MS pump) as described (26Cristea M. Oliw E.H. On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant.J. Lipid Res. 2007; 48: 890-903Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The combined effluents were introduced by electrospray into the ion trap mass spectrometer (LTQ). Chiral phase (CP)-HPLC was performed with Chiralcel OD (25 × 0.46 cm; Daicel through Skandinaviska GeneTech, Kungsbacka, Sweden) and eluted with 5% isopropanol in hexane with 0.1% acetic acid (0.5 ml/min). The effluent was analyzed by photodiode array detection, mixed with isopropanol-water (3:2; 0.3 ml/min), and subjected to MS/MS analysis of carboxylate anions.Miscellaneous11-HPOTrE was isolated from incubations of 20–200 ml of 0.1 M NaBO3 buffer (pH 9.0) with 100 μM 18:3n-3 and ∼4 nM Mn-LOX when the linear increase in UV absorbance started to deviate. The products were extracted with cold ethyl acetate, evaporated to dryness, and purified by RP-HPLC [methanol-water-acetic acid, 750:250:0.1; 4.2 × 100 mm, 5 μm Hypersil Gold (Thermo) or 10 μm, 8 × 300 mm, μBondapak C18 (Waters)], with UV detection at 210 and 237 nm. Fractions containing 11-HPOTrE were extracted with CH2Cl2 and evaporated to dryness, and the amount of 11-HPOTrE was estimated by UV analysis after conversion of an aliquot to 13-HPOTrE with Mn-LOX. 1-Linoleoyl-lysoGPC was obtained by treatment of dilinoleoyl-GPC with phospholipase A2, purified by preparative TLC (CHCl3/methanol/NH3/H2O, 6:3.6:0.4:0.4; Rf ∼ 0.23), and characterized by RP-HPLC-MS/MS analysis. Hydrolysis of oxygenated 1-linoleoyl-lysoGPC was performed with cholesteryl esterase as described (29Huang L.S. Kim M.R. Sok D-E. Linoleoyl lysophosphatidylcholine is an efficient substrate for soybean lipoxygenase-1.Arch. Biochem. Biophys. 2006; 455: 119-126Crossref PubMed Scopus (25) Google Scholar).RESULTS AND DISCUSSION18:3n-3 was rapidly oxidized by Mn-LOX, as shown by Fig. 1A, and the kinetic parameters (Km = 2.4 μM, Vmax = 17.6 μmol/min/mg, and kcat = 2,400 min−1) have been reported previously (14Su C. Oliw E.H. Manganese lipoxygenase. Purification and characterization.J. Biol. Chem. 1998; 273: 13072-13079Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). During the rapid linear phase of biosynthesis of UV light-absorbing products at 237 nm (13-HPOTrE, traces of 9-HPOTrE), the apparent amounts of the two main products were estimated by LC-MS/MS analysis. 11-HPOTrE averaged 25% (range, 22–27%), and 13-HPOTrE averaged ∼75%. The first linear increase in UV absorbance was followed by a second linear phase (at a rate of 13–15% of the first), during which 11-HPOTrE was converted to 13-HPOTrE (15Hamberg M. Su C. Oliw E. Manganese lipoxygenase. Discovery of a bis-allylic hydroperoxide as product and intermediate in a lipoxygenase reaction.J. Biol. Chem. 1998; 273: 13080-13088Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The rearrangement of 11-HPOTrE occurred slowly when low amounts of enzyme were used (4 nM Mn-LOX; Fig. 1A), which provided a practical way to generate 11-HPOTrE.Fig. 1Oxygenation of 18:3n-3 and 20:4n-6 by manganese lipoxygenase (Mn-LOX). A: Progression curve with ultraviolet (UV) and LC-MS/MS analysis of products formed during the oxygenation of 18:3n-3 by Mn-LOX. Mn-LOX (19, 7, and 4 nM) was incubated with 18:3n-3, aliquots were analyzed for 11-hydroperoxyoctadecadienoic acid (11-HPODE) and 13-HPODE, and the ratio 11-HPODE/(11-HPODE + 13-HPODE) is indicated at these time points. B: Oxidation of 20:4n-6 by Mn-LOX (72 nM). The bis-allylic intermediate 13-hydroperoxyeicosatetraenoic acid (13-HPETE) was rapidly transformed to 15-HPETE, as indicated by MS/MS analysis at two time points at the upper end of the progression curve.View Large Image Figure ViewerDownload Hi-res image Download (PPT)20:4n-6 is a poor substrate of Mn-LOX and required large amounts of enzyme for rapid transformation (25Cristea M. Oliw E.H. A G316A mutation of manganese lipoxygenase augments hydroperoxide isomerase activity: mechanism of biosynthesis of epoxyalcohols.J. Biol. Chem. 2006; 281: 17612-17623Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Cristea M. Oliw E.H. On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant.J. Lipid Res. 2007; 48: 890-903Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Oxidation of 20:4n-6 by Mn-LOX and analysis of the products are illustrated in Fig. 1B. Analysis of the products during the rapid and almost linear increase in UV absorbance at 235 nm, which is attributable mainly to the formation of 15R-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HPETE) (26Cristea M. Oliw E.H. On the singular, dual, and multiple positional specificity of manganese lipoxygenase and its G316A mutant.J. Lipid Res. 2007; 48: 890-903Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), showed that 13-hydroperoxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (13-HPETE) accounted for ∼10–14% of the products during the upper part of the linear phase. This number was below 1% already at the peak of UV absorbance and was <0.1% at later time points (data not shown). In comparison with 20:3n-3, the bis-allylic hydroperoxide of 20:4n-6 was rearranged rapidly.Oxygenation of dilinoleoyl-GPC suggested that fatty acids bind the active site of Mn-LOX tail-first (26Cristea M. Oliw E.H. On the singular, dua
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