The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size ...Read More
Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe390, Gly441, Ala455, and Val631 align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe390 and Ala455 did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly441 to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val631, which aligns with the positional determinant Ile593 of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val631 for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val631 as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products. Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe390, Gly441, Ala455, and Val631 align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe390 and Ala455 did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly441 to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val631, which aligns with the positional determinant Ile593 of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val631 for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val631 as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products. Lipoxygenases (LOXs) 2The abbreviations used are:LOXslipoxygenases(11R)-HETE(11R,5Z,8Z,12E,14Z)-11-hydroxy-5,8,12,14-eicosatetraenoic acid(12R)- and (12S)-HETEs(12R,5Z,8Z,10E,14Z)- and (12S,5Z,8Z,10E,14Z)-12-hydroxy-5,8,10,14-eicosatetraenoic acidsHPLChigh performance liquid chromatography(15R)- and (15S)-HETEs(15R,5Z,8Z,11Z,13E)- and (15S,5Z,8Z,11Z,13E)-15-hydroxy-5,8,11,13-eicosatetraenoic acids(8R)- and (8S)-HETEs(8R,5Z,9E,11Z,14Z)- and (8S,5Z,9E,11Z,14Z)-8-hydroxy-5,9,11,14-eicosatetraenoic acids(5R)- and (5S)-HETE(5R,6E,8Z,11Z,14Z)- and (5S,6E,8Z,11Z,14Z)-5-hydroxy-6,8,11,14-eicosatetraenoic acidsPBSphosphate-buffered salineRPreverse-phaseSPstraight-phaseCPchiral-phaseGC/MSgas chromatography/mass spectrometrydiHETEdihydroxyeicosatetraenoic acidHODEhydroxyoctadecadienoic acid. form a heterogeneous family of lipid peroxidizing enzymes that catalyze dioxygenation of free and/or esterified polyunsaturated fatty acids to their corresponding hydroperoxy derivatives (1Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar). They are involved in the biosynthesis of eicosanoids (2Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3070) Google Scholar) such as the pro-inflammatory leukotrienes (3Samuelsson B. Dahlen S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1987) Google Scholar) and anti-inflammatory lipoxins (4Serhan C.N. Levy B. Chem. Immunol. Allergy. 2003; 83: 115-145Crossref PubMed Scopus (38) Google Scholar), but have also been implicated in cell maturation (5van Leyen K. Duvoisin R.M. Engelhardt H. Wiedmann M. Nature. 1998; 395: 392-395Crossref PubMed Scopus (255) Google Scholar), cancer (6Pidgeon G.P. Kandouz M. Meram A. Honn K.V. Cancer Res. 2002; 62: 2721-2727PubMed Google Scholar), psoriasis (7Hammarström S. Hamberg M. Samuelsson B. Duell E.A. Stawisky M. Voorhees J.J. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 5130-5134Crossref PubMed Scopus (505) Google Scholar), atherogenesis (8Cathcart M.K. Folcik V.A. Free Radic. Biol. Med. 2000; 28: 1726-1734Crossref PubMed Scopus (98) Google Scholar), and osteoporosis (9Klein R.F. Allard J. Avnur Z. Nikolcheva T. Rotstein D. Carlos A.S. Shea M. Waters R.V. Belknap J.K. Peltz G. Orwoll E.S. Science. 2004; 303: 229-232Crossref PubMed Scopus (251) Google Scholar). lipoxygenases (11R,5Z,8Z,12E,14Z)-11-hydroxy-5,8,12,14-eicosatetraenoic acid (12R,5Z,8Z,10E,14Z)- and (12S,5Z,8Z,10E,14Z)-12-hydroxy-5,8,10,14-eicosatetraenoic acids high performance liquid chromatography (15R,5Z,8Z,11Z,13E)- and (15S,5Z,8Z,11Z,13E)-15-hydroxy-5,8,11,13-eicosatetraenoic acids (8R,5Z,9E,11Z,14Z)- and (8S,5Z,9E,11Z,14Z)-8-hydroxy-5,9,11,14-eicosatetraenoic acids (5R,6E,8Z,11Z,14Z)- and (5S,6E,8Z,11Z,14Z)-5-hydroxy-6,8,11,14-eicosatetraenoic acids phosphate-buffered saline reverse-phase straight-phase chiral-phase gas chromatography/mass spectrometry dihydroxyeicosatetraenoic acid hydroxyoctadecadienoic acid. Mechanistically, the LOX reaction consists of four elementary reactions, the stereochemistry of which is tightly controlled (see Scheme 1): (i) stereoselective hydrogen abstraction from a bisallylic methylene, forming a carbon-centered fatty acid radical; (ii) [+2] or [–2] rearrangement of the fatty acid radical; (iii) stereospecific insertion of molecular dioxygen, forming an oxygen-centered hydroperoxy radical; and (iv) reduction of the hydroperoxy fatty acid radical to the corresponding anion. Our current understanding of how mammalian LOXs control the stereochemistry of the oxygenation reaction is derived from the x-ray structure of rabbit reticulocyte 15-LOX (10Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar), from extensive mutagenesis studies on various LOX isoforms (11Sloane D.L. Leung R. Craik C.S. Sigal E. Nature. 1991; 354: 149-152Crossref PubMed Scopus (183) Google Scholar, 12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar, 13Suzuki H. Kishimoto K. Yoshimoto T. Yamamoto S. Kanai F. Ebina Y. Miyatake A. Tanabe T. Biochim. Biophys. Acta. 1994; 1210: 308-316Crossref PubMed Scopus (64) Google Scholar, 14Watanabe T. Haeggström J.Z. Biochem. Biophys. Res. Commun. 1993; 192: 1023-1029Crossref PubMed Scopus (40) Google Scholar, 15Jisaka M. Kim R.B. Boeglin W.E. Brash A.R. J. Biol. Chem. 2000; 275: 1287-1293Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and from experiments with chemically modified fatty acid substrates (16Kühn H. Sprecher H. Brash A.R. J. Biol. Chem. 1990; 265: 16300-16305Abstract Full Text PDF PubMed Google Scholar, 17Walther M. Ivanov I. Myagkova G. Kuhn H. Chem. Biol. 2001; 115: 1-13Google Scholar). The substrate-binding cleft of the rabbit enzyme is a U-shaped pocket, the bottom of which is defined by a triad of amino acids (Phe353, Ile418, and Ile593). A simple model for substrate alignment at the active site of this enzyme suggests that polyenoic fatty acids may slide into the substrate-binding pocket with their methyl end ahead (12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar). Molecular modeling (10Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar, 18Browner M.F. Gillmor S.A. Fletterick R. Nat. Struct. Biol. 1998; 5: 179Crossref Scopus (34) Google Scholar) and site-directed mutagenesis (12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar) suggest that the volume of the active site might be important for the positional specificity. This space-related hypothesis was initially opposed by the orientation-based model, which suggests the possibility of an inverse head-to-tail substrate alignment (19Gardner H.W. Biochim. Biophys. Acta. 1989; 1001: 274-281Crossref PubMed Scopus (176) Google Scholar, 20Prigge S.T. Gaffney B.J. Amzel L.M. Nat. Struct. Biol. 1998; 5: 178Crossref PubMed Scopus (33) Google Scholar). However, more recent experimental data suggest that both hypotheses appear to be valid (12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar, 17Walther M. Ivanov I. Myagkova G. Kuhn H. Chem. Biol. 2001; 115: 1-13Google Scholar). Most of the mechanistic studies performed out in the past on the structural basis for the positional specificity of LOXs have been carried out on classical S-LOX isoforms (see Ref. 12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar for review), but little is known about the corresponding mechanisms of the more recently discovered R-lipoxygenating enzyme species (21Boeglin W.E. Kim R.B. Brash A.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6744-6749Crossref PubMed Scopus (147) Google Scholar, 22Krieg P. Siebert M. Kinzig A. Bettenhausen R. Marks F. Fürstenberger G. FEBS Lett. 1999; 446: 142-148Crossref PubMed Scopus (42) Google Scholar, 23Brash A.R. Boeglin W.E. Chang M.S. Shieh B.H. J. Biol. Chem. 1996; 271: 20949-20955Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). However, during the preparation of this manuscript, a study that also included two R-LOXs was published (24Coffa G. Brash A.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 44: 12579-15584Google Scholar). Multiple amino acid sequence alignments of R- and S-LOXs suggest that the R-lipoxygenating enzymes contain a conserved Gly in the central part of their primary structure. Mutation of Gly427 to Ala in (8R)-LOX from corals induces alterations in the positional specificity as well as changes in the enantioselectivity of the enzyme (predominant formation of (12S)-HETE methyl ester). Similar modifications were observed when corresponding mutations were carried out with human (12R)-LOX, murine (8S)-LOX, and human (15S)-LOX2. To investigate the structural basis for the stereochemical control mechanisms of murine (12R)-LOX more comprehensively, we applied a strategy that involves targeted substrate modification and site-directed mutagenesis. For this purpose, we first identified putative sequence determinants for the reaction specificity by multiple amino acid sequence alignments and modified their side chains. The data obtained indicate that mutation of Ala455 and Phe359 led to the formation of enzyme species with reduced specific activities and stereo random product patterns. In contrast, introduction of less bulky residues at Val631 induced both an increase in catalytic activities and specific alterations in product patterns, with (11R)-HETE methyl ester being the major oxygenation product. These data indicate that Val631 constitutes a major sequence determinant for both the positional specificity and enantioselectivity of murine (12R)-LOX because the geometry of its side chain impacts the stereochemistry of both initial hydrogen abstraction and subsequent oxygen insertion. Chemicals—The chemicals used were purchased from the following sources: arachidonic acid methyl ester (5Z,8Z,11Z,14Z-eicosatetraenoic acid methyl ester) and isopropyl β-d-thiogalactopyranoside from Sigma (Deisenhofen, Germany); HPLC standards of (12R)- and (12S)-HETEs, (15R)- and (15S)-HETEs, (11R)- and (11S)-HETEs, (8R)- and (8S)-HETEs, (9R,5Z,7E,11Z,14Z)- and (9S,5Z,7E,11Z,14Z)-9-hydroxy-5,7,11,14-eicosatetraenoic acids, and (5R)- and (5S)-HETEs from Cayman Chemical Co. Inc. (Ann Arbor, MI); sodium borohydride from Serva (Heidelberg, Germany); ampicillin from Invitrogen (Eggenstein, Germany); and HPLC solvents from Merck (Darmstadt, Germany). Restriction enzymes were purchased from New England Biolabs Inc. (Schwalbach, Germany). The QuikChange site-directed mutagenesis kit was from Stratagene (La Jolla, CA). The BaculoGold™ transfection kit and Sf9 insect cells were from BD Biosciences. Oligonucleotide synthesis was carried out by TIB MOLBIOL (Berlin, Germany). All other chemicals and solvents were of analytical grade. Expression of (12R)-LOX—Murine (12R)-LOX was expressed in Escherichia coli as an N-terminally His-tagged fusion protein. For this purpose, the coding region of the cDNA was ligated into the pQE expression vector, and bacteria were transformed with the recombinant plasmid containing an ampicillin resistance gene as selection marker. Bacteria were cultured at 37 °C in 1 liter of LB medium containing 0.1 mg/ml ampicillin to reach an absorbance at 600 nm of 0.5. The expression of the recombinant protein was induced by addition of isopropyl β-d-thiogalactopyranoside (1 mm final concentration). After 2 h of incubation, bacteria were spun down, washed twice, resuspended in 20 ml of phosphate-buffered saline (PBS), and lysed by sonication. Cell debris was removed by centrifugation, and the clear lysis supernatant was used as enzyme source. Activity assays were performed by addition of variable amounts of lysis supernatant to 0.5 ml of PBS containing arachidonic acid methyl ester (0.1 mm final concentration) as substrate. The mixture was incubated for 15 min at 37 °C, and the hydroperoxy lipids formed were reduced to the more stable hydroxy derivatives by addition of 0.1 ml of saturated solution of sodium borohydride in dry ethanol. After acidification to pH 3 (acetic acid), 0.5 ml of methanol was added, and the samples were kept on ice for 10 min. The protein precipitate was spun down, and aliquots of the clear supernatant were directly analyzed by reverse-phase (RP) HPLC for quantification of the LOX products. Selected mutants of murine (12R)-LOX were expressed in Sf9 insect cells using the BaculoGold™ transfection system. For this purpose, the corresponding wild-type or mutant cDNA was ligated into the pVL1392 transfer vector containing a His tag at the N terminus of the enzyme. Highly purified recombinant transfer vectors were used for cotransfection of insect cells together with wild-type baculovirus. For this purpose, the recombinant transfer vector (5 μg) was mixed with 1 μg of BaculoGold™ DNA and added to a monolayer of Sf9 insect cells (2 × 106 cells/well) in TNM-FH medium. After 72 h, the culture supernatant containing recombinant baculovirus was used to re-infect freshly cultured Sf9 cells to produce high titer virus stocks. Finally, the high titer virus stock was diluted with culture medium to a multiplicity of infection of 5 and was used for infecting freshly cultured Sf9 cells (106 cells/ml). 3 days after infection, the cells were spun down, reconstituted in PBS, and sonicated with a tip sonifier (Braun, Melsungen, Germany). Cell debris was removed by centrifugation, and the clear lysis supernatant was used as enzyme source. Mutagenesis—Site-directed mutagenesis was performed using the QuikChange mutagenesis kit following the manufacturer's instructions. To identify mutant LOX clones, 10–20 clones were selected and screened for the mutations by restriction mapping and activity assays. Finally, mutations were confirmed by sequencing. Analytics—HPLC was performed on a Shimadzu HPLC system connected to a Hewlett-Packard 1040A diode array detector. RP-HPLC was carried out on a Nucleosil C18 column (KS system, 250 × 4 mm, 5-μm particle size; Macherey Nagel, Duren, Germany) coupled with a guard column (30 × 4 mm, 5-μm particle size). A solvent system of methanol/water/acetic acid (85:15:0.1 by volume) was used at a flow rate of 1 ml/min. Straight-phase (SP) HPLC was performed on a Zorbax SIL column (250 × 4 mm, 5-μm particle size) with a solvent system of n-hexane/2-propanol/acetic acid (100:2:0.1 by volume) at a flow rate of 1 ml/min. For chiral-phase (CP) HPLC, we used a Chiralcel OD or a Chiralcel OB column (250 × 4 mm, 5-μm particle size) and a solvent system consisting of n-hexane/acetic acid (100:0.1 by volume) containing 2-propanol in various concentrations depending on the chemistry of the reaction products. The flow rate was 1 ml/min. Gas Chromatography/Mass Spectrometry (GC/MS)—GC/MS was carried out on a Shimadzu GC/MS QP-2000 system equipped with a fused SPB1 silica column (10 m × 0.25 mm, 0.25-μm coating thickness) at an injector temperature of 270 °C. An ion source temperature of 180 °C and an electron energy of 70 eV were adjusted. The derivatized fatty acids were eluted with the following temperature program: isothermal run at 180 °C for 2 min and then from 180 to 290 °C at a rate of 5 °C/min. Miscellaneous Methods—Protein concentrations were determined using the Roti-Quant kit (Carl Roth GmbH, Karlsruhe, Germany). Methylation of free carboxylic acids was achieved by bubbling ethereal diazomethane with argon (2 min) and transferring the diazomethane gas to the fatty acid solution. For more informative mass spectra, the methylated derivatives of the polyenoic fatty acids (10 μg dissolved in 1 ml of methanol) were hydrogenated using 5 mg of 10% palladium/CaCO3 (Merck) as catalyst. Hydrogen gas was bubbled through this mixture for 2 min at room temperature. The solution was filtered to remove the catalyst; the solvent was evaporated; and the products were silylated. Aliquots (2 μl) were injected into the GC/MS system. Km values for the formation of the different oxygenation products were determined by incubating the enzyme preparations with different concentrations of arachidonic acid methyl ester (7–100 μm) and quantifying the different HETE isomers separately by RP-HPLC. 8- and 9-HETEs were not well separated under our experimental conditions; and thus, the sum of both products was evaluated. Mutagenesis of Positional Determinants Identified for Other LOX Isoforms—Previous mutagenesis studies on human and rabbit reticulocyte 15-LOXs identified Ile418 and Met419 and Phe353 and Ile593, respectively, as sequence determinants for the positional specificity (11Sloane D.L. Leung R. Craik C.S. Sigal E. Nature. 1991; 354: 149-152Crossref PubMed Scopus (183) Google Scholar, 12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar), but no information is currently available on the importance of these residues for the specificity of murine (12R)-LOX. To test the relevance of these amino acids, we first performed multiple sequence alignments. Fig. 1A shows that Phe353 of rabbit 15-LOX aligns with Phe390 of murine (12R)-LOX. Similarly, Ile418 and Ile593 of the rabbit enzyme align with Ala455 and Val631 of the murine enzyme, respectively (Fig. 1, B and C). Because Ile is more space-filling than Ala and Val, one may conclude that murine (12R)-LOX may have a bigger substrate-binding pocket than rabbit 15-LOX. If the volume of the substrate-binding pocket is important for the positional specificity of murine (12R)-LOX, introduction of bulkier residues at these positions should alter the enzyme specificity in favor of C-13 hydrogen removal. In this case, 15- and/or 11-HETE methyl ester should be the major oxygenation product. To test this hypothesis, we first expressed murine (12R)-LOX in E. coli and then mutated Ala455 to a somewhat bulkier residue (Ile). TABLE ONE shows that this mutant exhibited a strongly reduced specific activity and a more random positional specificity compared with the wild-type enzyme. C-13 hydrogen abstraction was increased from 18% (wild-type enzyme) to 43% (A455I). Between the two positional isomers originating from C-13 hydrogen removal (15- and 11-HETEs), the formation of 11-HETE methyl ester, which involves [–2] rearrangement of the fatty acid radical, was dominant (33% of the sum of HETE isomers). Interestingly, the 11-HETE methyl ester formed was chiral (R/S ratio of 98:2), and a similar R/S ratio was analyzed for 12-HETE. 15-HETE methyl ester contributed only ∼10% to the product mixture. To determine whether the alterations induced by the A455I mutation will become more pronounced when an even bulkier residue is introduced, we created the A455W mutant. This mutant was also less active than the wild-type enzyme, and C-10 hydrogen abstraction was dominant (50%). (12R)-HETE methyl ester was the major oxygenation product (TABLE ONE), and an R/S ratio of 86:14 was determined. In addition, this mutant catalyzed significant C-7 hydrogen abstraction, resulting in the formation of similar amounts of 9- and 5-HETE methyl esters. Next, we thought about introducing a less space-filling Gly at position 455. However, sequence alignment with human (12R)-LOX indicated that this isoform has a Gly at this position; and thus, no major impact of the A455G mutation was expected. Summarizing these data, one may conclude that introduction of more space-filling residues at Ala455 impairs both the catalytic activity and positional specificity of the enzyme. The alterations in positional specificity can hardly be explained on the basis of the space-related hypothesis (10Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar, 18Browner M.F. Gillmor S.A. Fletterick R. Nat. Struct. Biol. 1998; 5: 179Crossref Scopus (34) Google Scholar) regardless of whether a straight (methyl end first) or an inverse (carboxylate first) substrate orientation might be involved.TABLE ONEImpact of site-directed mutagenesis of putative specificity determinants on the specific activity and positional specificity of murine (12R)-LOXProduct compositionEnzymeImpact on active-site volumeSpecific activityC-13 hydrogen abstractionC-10 hydrogen abstractionC-7 hydrogen abstraction15-HETE11-HETE12-HETE8-HETE9-HETE5-HETE%%Wild-type10001863 (92:8)1270A4551æ101033 (98:2)2313210A455Wâ4581233 (86:14)171416F390Wæ2NDNDNDNDNDNDF390Aá5081429 (86:14)171220V631Fâ4NDNDNDNDNDNDV631Aä250249 (99:1)1416190V631Gá273242 (98:2)1315290 Open table in a new tab To test the possible importance of Phe390 (12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar) for the positional specificity of murine (12R)-LOX, we first mutated Phe390 to a more space-filling residue (Trp). Unfortunately, this mutant had almost no activity (TABLE ONE); and thus, no product analysis was carried out. Next, the bulky Phe390 was mutated to a less space-filling residue (Ala). This mutant exhibited a reduced specific activity, and we observed a complex mixture of oxygenation products involving all possible positional isomers (TABLE ONE). As for the wild-type enzyme, 12-HETE (R/S ratio of 86:14) was identified as the major oxygenation product. The crystal structure of rabbit 15-LOX predicted the importance of Ile593 for the positional specificity (10Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (395) Google Scholar), and site-directed mutagenesis studies confirmed this prediction (12Kuhn H. Prostaglandins Other Lipid Mediat. 2000; 62: 255-270Crossref PubMed Scopus (71) Google Scholar). To reduce the volume of the active site of murine (12R)-LOX, we next introduced bulkier residues at this position. When Val631 of murine (12R)-LOX was mutated to an amino acid with a larger side chain (Ile or Phe), a graded reduction of the specific activity was observed (residual activities of 24 and 4% for the V631I and V631F mutants, respectively). Moreover, these mutants exhibited a strongly impaired positional specificity (data not shown). Finally, we mutated Val631 to an amino acid carrying a smaller side chain (Ala or Gly) to increase the volume of the substrate-binding pocket. Here, we observed a strong increase in the catalytic activity and more specific alterations in the reaction specificity. For both mutants, we identified (11R)-HETE as the major oxygenation product. In contrast, 12-HETE methyl ester was strongly reduced (TABLE ONE). In E. coli, murine (12R)-LOX was expressed at only low levels, and purification of the recombinant enzyme was not possible. For more detailed investigations, the wild-type enzyme and the most interesting mutants (V631A and V631G) were overexpressed as His-tagged fusion proteins in the baculovirus/insect cell system and purified on a nickel-agarose column. The enzyme expressed in insect cells exhibited a 4-fold higher specific activity with methyl linoleate as substrate (10 μg of HETE/μg of enzyme in E. coli versus 38 μg of HETE/μg of enzyme in insect cells), and similar alterations were observed in the methyl arachidonate system. Compared with the wild-type enzyme, the V631A mutation induced a 6.6-fold increase in methyl linoleate oxygenase activity. For the V631G mutant, the increase was ∼5.6-fold. The alterations in the product pattern observed for the “bacterial enzyme” were confirmed in the insect cell system. Here again, 11-HETE methyl ester was identified as the major oxygenation product for the V631A mutant (Fig. 2A), and analysis of the enantiomer composition indicated a strong preponderance of the R-isomer (Fig. 2C). The 8-HETE methyl ester formed by the mutant enzyme was also chiral, but the S-enantiomer was dominant (Fig. 2C). This finding contrasts with the stereochemistry of the 8-HETE formed by the wild-type enzyme (Fig. 2B). These data suggest that, for the formation of 8-HETE, the substrate fatty acids might be aligned differently at the active site of the two enzyme species. Summarizing these data, one concludes that the V631A and V631G mutations lead to a strong increase in the catalytic activity and to major changes in the product specificity. Interestingly, for 12R- and 11R-lipoxygenation, oxygen insertion proceeds from opposite sides of the double bond system (see Scheme 2A). These alterations can be understood if formation of these oxygenation products involves a variable substrate alignment at the enzyme. When a substrate adopts multiple catalytically productive conformations at the active site, the binding kinetics should be different. To test this possibility, we determined the KM values for the formation of (12R)- and (11R)-HETEs by the V631A mutant. Fig. 3 (a Lineweaver-Burk plot) shows that the substrate affinity for (11R)-HETE formation (KM = 10.8 μm) was significantly higher than that for (12R)-HETE formation (KM = 15.8 μm). Nonlinear least-square fitting of our experimental data to the Michaelis-Menten equation revealed similar kinetic constants (KM = 13.7 μm for (11R)-HETE and KM = 24.1 μm for (12R)-HETE). These data suggest a different substrate alignment at the active site for the formation of (12R)- and (11R)-HETEs, and this conclusion is consistent with our interpretation of the mechanistic reasons for the different positional specificities of the two enzyme species (see Scheme 2B).FIGURE 3Kinetic measurements of arachidonic acid methyl ester oxygenation by the V631A mutant of murine (12R)-LOX. The mutant enzyme (purified enzyme preparation) was incubated with different concentrations of arachidonic acid methyl ester (7–100 μm). After 15 min, the incubation was stopped, and the formation of 12-, 11-, and 9/8-HETEs was quantified separately by RP-HPLC (see “Experimental Procedures”). 8- and 9-HETEs were not well separated under our experimental conditions; and thus, the sum of both products was used for evaluation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reactivity with the Mechanistic Probe 20-HETE Methyl Ester—When rabbit 15-LOX reacts with methyl arachidonate, formation of (15S)-HETE methyl ester is dominant; in contrast, when 20-HETE methyl ester is used as substrate, 5-lipoxygenation prevails, and an inverse head-to-tail substrate orientation was suggested (25Ivanov I. Rathmann J. Myagkova G. Kuhn H. Biochemistry. 2001; 40: 10223-10229Crossref PubMed Scopus (9) Google Scholar). To test whether or not 20-HETE methyl ester is oxygenated by murine (12R)-LOX with a different positional specificity compared with methyl arachidonate, we incubated the wild-type enzyme and its V631A mutant with 20-HETE met
