Secretion of two novel enzymes, manganese 9S-lipoxygenase and epoxy alcohol synthase, by the rice pathogen Magnaporthe salvinii
2012; Elsevier BV; Volume: 54; Issue: 3 Linguagem: Inglês
10.1194/jlr.m033787
ISSN1539-7262
AutoresAnneli Wennman, Ernst H. Oliw,
Tópico(s)Biochemical and biochemical processes
ResumoThe mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion. The mycelium of the rice stem pathogen, Magnaporthe salvinii, secreted linoleate 9S-lipoxygenase (9S-LOX) and epoxy alcohol synthase (EAS). The EAS rapidly transformed 9S-hydroperoxy-octadeca-10E,12Z-dienoic acid (9S-HPODE) to threo 10 (11)-epoxy-9S-hydroxy-12Z-octadecenoic acid, but other hydroperoxy FAs were poor substrates. 9S-LOX was expressed in Pichia pastoris. Recombinant 9S-LOX oxidized 18:2n-6 directly to 9S-HPODE, the end product, and also to two intermediates, 11S-hydroperoxy-9Z,12Z-octadecenoic acid (11S-HPODE; ∼5%) and 13R-hydroperoxy-9Z,11E-octadecadienoic acid (13R-HPODE; ∼1%). 11S- and 13R-HPODE were isomerized to 9S-HPODE, probably after oxidation to peroxyl radicals, β-fragmentation, and oxygen insertion at C-9. The 18:3n-3 was oxidized at C-9, C-11, and C-13, and to 9,16-dihydroxy-10E,12,14E-octadecatrienoic acid. 9S-LOX contained catalytic manganese (Mn:protein ∼0.2:1; Mn/Fe, 1:0.05), and its sequence could be aligned with 77% identity to 13R-LOX with catalytic manganese lipoxygenase (13R-MnLOX) of the Take-all fungus. The Leu350Met mutant of 9S-LOX shifted oxidation of 18:2n-6 from C-9 to C-13, and the Phe347Leu, Phe347Val, and Phe347Ala mutants of 13R-MnLOX from C-13 to C-9. In conclusion, M. salvinii secretes 9S-LOX with catalytic manganese along with a specific EAS. Alterations in the Sloane determinant of 9S-LOX and 13R-MnLOX with larger and smaller hydrophobic residues interconverted the regiospecific oxidation of 18:2n-6, presumably by altering the substrate position in relation to oxygen insertion. atomic emission spectroscopy buffered glycerol complex medium buffered minimal methanol chiral phase dihydroxyoctadecatrienoic acid epoxy alcohol synthase hydroxyoctadecadienoic acid hydroxyoctadecatrienoic acid hydroperoxyoctadecadienoic acid hydroperoxyoctadecamonoenoic acid hydroperoxyoctadecatrienoic acid linoleate diol synthase lipoxygenase manganese lipoxygenase reverse phase soybean lipoxygenase-1 ultraviolet Lipoxygenases (LOXs) oxidize polyunsaturated FAs to hydroperoxides, which can be further transformed to an array of biological mediators, e.g., eicosanoids and leukotrienes in man and to oxylipins and jasmonates in plants and fungi (1Haeggström J.Z. Funk C.D. 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(2009) Magneporthe and its relatives. In, Encylcopedia of Life Sciences. John Wiley & Sons, Ltd, Chichester.Google Scholar). Oxylipin biosynthesis by G. graminis and M. oryzae has been studied (4Brodhun F. Feussner I. Oxylipins in fungi.FEBS J. 2011; 278: 1047-1063Crossref PubMed Scopus (143) Google Scholar, 19Ribot C. Hirsch J. Balzergue S. Tharreau D. Notteghem J.L. Lebrun M.H. Morel J.B. Susceptibility of rice to the blast fungus, Magnaporthe grisea.J. Plant Physiol. 2008; 165: 114-124Crossref PubMed Scopus (139) Google Scholar), but little is known about oxylipin biosynthesis by M. salvinii. The latter infects the stem and the leaf sheats at the water line, leading to necrosis and formation of small black sclerotia with conidia (16Besi, M. I., Tucker, S. L., Sesma, A., . (2009) Magneporthe and its relatives. In, Encylcopedia of Life Sciences. John Wiley & Sons, Ltd, Chichester.Google Scholar, 20Krause R.A. Webster R.K. Morphology, taxonomy, and sexuality of rice stem rot fungus, Magnaporthe Salvinii (Leptosphaeria Salvinii).Mycologia. 1972; 64: 103-114Crossref Google Scholar). The LOX family is characterized by sequence homology with conserved ligands to the catalytic nonheme iron (2Andreou A. Feussner I. Lipoxygenases - Structure and reaction mechanism.Phytochemistry. 2009; 70: 1504-1510Crossref PubMed Scopus (296) Google Scholar, 5Kuhn H. Saam J. Eibach S. Holzhutter 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 (99) Google Scholar, 21Schneider C. Pratt D.A. Porter N.A. Brash A.R. Control of oxygenation in lipoxygenase and cyclooxygenase catalysis.Chem. Biol. 2007; 14: 473-488Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). These octahedral ligands are three His residues, the carboxyl group of the C-terminal amino acid, usually Ile, a distant Asn or His residue, and water. The latter forms the catalytic base for hydrogen abstraction at bis-allylic carbons of FAs and subsequent oxidation. In this process of proton-coupled electron transfer, the catalytic base (Fe3+OH−) is reduced (Fe2+OH2), and oxygen is inserted in an antarafacial manner at either C-1, C-5, or C-3 of the 1,4-pentadiene radical with formation of peroxyl radicals (21Schneider C. Pratt D.A. Porter N.A. Brash A.R. Control of oxygenation in lipoxygenase and cyclooxygenase catalysis.Chem. Biol. 2007; 14: 473-488Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 22Hamberg 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 (116) Google Scholar). In the next step, hydrogen is transferred to the peroxyl radicals with regeneration of the catalytic base, Fe3+OH−. Iron was identified as the catalytic metal of soybean LOX-1 (sLOX-1) (23Chan H.W.-S. Soya-bean lipoxygenase: an iron-containing dioxygenase.Biochim. Biophys. Acta. 1973; 327: 32-35Crossref PubMed Scopus (89) Google Scholar, 24Pistorius E.K. Axelrod B. Iron, an essential component of lipoxygenase.J. Biol. Chem. 1974; 249: 3183-3186Abstract Full Text PDF PubMed Google Scholar), and later in many other LOXs. To date, the only known exception is 13R-MnLOX. A comparison of 13R-MnLOX with FeLOXs revealed three principal differences. First, the primary sequences of the catalytic domain of 13R-MnLOX contains a pentamer motif flanked by two His metal ligands, His-Val-Leu-Phe-His, in the presumed manganese binding region (25Hörnsten 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 (39) Google Scholar, 26Cristea 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 corresponding two His ligands of FeLOX are found in a conserved hexamer, His-(Leu/Trp)-Leu-(Asn/Arg)-(Thr/Gly)-His (Fig. 1A). Second, 13R-MnLOX catalyzes suprafacial hydrogen abstraction and oxygenation (22Hamberg 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 (116) Google Scholar). Third, 13R-MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals about two orders of magnitude more rapidly than sLOX-1 and other FeLOXs (27Oliw E.H. Factors influencing the rearrangement of bis-allylic hydroperoxides by manganese lipoxygenase.J. Lipid Res. 2008; 49: 420-428Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 28Oliw E.H. Jernerén F. Hoffmann I. Sahlin M. Garscha U. Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms.Biochim. Biophys. Acta. 2011; 1811: 138-147Crossref PubMed Scopus (34) Google Scholar). To date, little is known about iron and manganese selection by LOXs. In pursuit of additional LOXs with catalytic manganese, we found that 9-LOX of M. salvinii (GenBank, CAD61974.1) contains the tentative manganese binding pentamer motif, His-Val-Leu-Phe-His. This sequence could be aligned to 13R-MnLOX (GenBank, AAK81882.1) with 77% amino acid identity and contains conserved structural elements also found in mammalian LOXs (Fig. 1A, B, and supplementary Fig. I). The tentative Mn ligands of 13R-MnLOX are conserved in the LOX of M. salvinii except for the exchange of the C-terminal Val residue to Ile, which is the predominant C-terminal amino acid of LOXs (Fig. 1A). Both enzymes contain Gly in the Coffa-Brash (Gly/Ala) determinant of regiospecificity and R/S chirality (29Coffa G. Brash A.R. A single active site residue directs oxygenation stereospecificity in lipoxygenases: stereocontrol is linked to the position of oxygenation.Proc. Natl. Acad. Sci. USA. 2004; 101: 15579-15584Crossref PubMed Scopus (154) Google Scholar), but they differ at one homologous position (Phe347/Leu350) of the Sloane determinant (30Sloane D.L. Leung R. Craik C.S. Sigal E. A primary determinant for lipoxygenase positional specificity.Nature. 1991; 354: 149-152Crossref PubMed Scopus (182) Google Scholar) (Fig. 1B). An overview of hydrogen abstraction and oxygenation of 18:2n-6 by LOXs is shown in Fig. 1C. Our first goal was to determine the oxylipin biosynthesis by M. salvinii and whether LOXs were secreted in analogy with G. graminis. We found that M. salvinii secreted linoleate 9S-LOX and a novel epoxy alcohol synthase (EAS) with specificity for 9S-hydroperoxy-10E,12Z-octadecadienoic acid (9S-HPODE). This enzyme was apparently identical to the recombinant 9-LOX of M. salvinii, which was described and patented by Sugio and Takagi at Novozymes (31Sugio, A., Takagi, S., . (2004) Variants of lipoxygenase and their use (EP1383874 A1). In Espacenet. Novozymes, Denmark.Google Scholar) with a detailed patent description available on the Internet 2For a detailed description of the patent of 9-LOX, see http://www.google.com/patents/EP1383874A1?cl=en. The patent describes cloning, expression, and characterization of recombinant 9-LOX of M. salvinii; its reaction with C18 and C20 PUFAs and esters; and its pH dependence, stability, and activity at elevated temperatures. For the corresponding patent of 13R-MnLOX, see EP1317528 (A2). 2For a detailed description of the patent of 9-LOX, see http://www.google.com/patents/EP1383874A1?cl=en. The patent describes cloning, expression, and characterization of recombinant 9-LOX of M. salvinii; its reaction with C18 and C20 PUFAs and esters; and its pH dependence, stability, and activity at elevated temperatures. For the corresponding patent of 13R-MnLOX, see EP1317528 (A2).. Our second goal was to determine the catalytic metal of this 9-LOX. To this end, we expressed 9S-LOX of M. salvinii in Pichia pastoris. The recombinant enzyme was found to oxidize 18:2n-6 and 18:3n-3 in the same way as native 9S-LOX and to contain Mn; it will therefore be referred to as 9S-MnLOX. Our third goal was to investigate the structural basis of the regiospecificities of 9S- and 13R-MnLOX. Finally, we studied the metal selection process of 13R-MnLOX by insertion of an amino acid (Thr/Gly) between the fourth and fifth positions of the His-Val-Leu-Phe-His pentamer motif and by expression in the environment of augmented intracellular iron concentration. HPLC solvents (Lichrosolve), 18:3n-6 (99%), and routine chemicals were from Merck or Sigma-Aldrich. The 18:2n-6 (99%), Mn(III) acetate 2-hydrate (98%), and polymer beads with iminodiacetic groups (Chelex-100), yeast nitrogen base, carbonic anhydrase, cytochrome C, α-mannosidase, and the TRI reagent were from Sigma-Aldrich. The 18:3n-3 (99%) and [13C18]18:2n-6 (98%) were from Larodan. [11S-2H]18:2n-6 (>95% 2H) and [11R-2H]18:2n-6 (∼28% 2H) were prepared as described (32Hamberg M. Stereochemistry of hydrogen removal during oxygenation of linoleic acid by singlet oxygen and synthesis of 11(S)-deuterium-labeled linoleic acid.Lipids. 2011; 46: 201-206Crossref PubMed Scopus (15) Google Scholar). FAs were dissolved in ethanol and stored in stock solutions (30–100 mM) at −20°C. Methyl 10(11)-epoxy-9S-hydroxy-12Z-octadecenoate (threo and erythro isomers; hydrolyzed to free acids with rat plasma) and 9(R/S)-hydroxy-10E,12E-octadecadienoic acid [(10E,12E)9(R,S)-HODE] were from Lipidox. Epoxy alcohols were prepared by hematin-catalyzed transformation of 9S-HPODE and by epoxidation of 13S-HODE and 11S-HODE with m-chloroperoxybenzoic acid (85%, Sigma) as described (33Oliw E.H. Garscha U. Nilsson T. Cristea M. Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry.Anal. Biochem. 2006; 354: 111-126Crossref PubMed Scopus (46) Google Scholar). 11R-hydroperoxy-9Z,12Z,15Z-octadecatrienoic acid (11R-HPOTrE) and 11S-HPODE were obtained by biosynthesis and isolated as described (28Oliw E.H. Jernerén F. Hoffmann I. Sahlin M. Garscha U. Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms.Biochim. Biophys. Acta. 2011; 1811: 138-147Crossref PubMed Scopus (34) Google Scholar). 13S-HPODE, 13R-HPODE, 13R-HPOTrE, 9S-HPODE, and 9S-HPOTrE were obtained by biosynthesis (sLOX-1, 13R-MnLOX, tomato LOX) and purified by reverse phase (RP)-HPLC or by open-column silicic acid chromatography. Racemic [13C18]HPODE was obtained by photooxidation of [13C18]18:2n-6, and racemic HPOTrE by autoxidation of 18:3n-3. 9S-hydroperoxy-10E-octadecenoic acid (9S-HPOME) was obtained as described (34Oliw E.H. Wennman A. Hoffmann I. Garscha U. Hamberg M. Jernerén F. Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases.J. Lipid Res. 2011; 52: 1995-2004Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Oxygen-18 (97%) was from Isotec/Sigma-Aldrich, and was used to prepare [18O]13R-HPODE (∼95% 18O incorporation) with 13R-MnLOX as described (22Hamberg 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 (116) Google Scholar). Columns with Reprosil Chiral NR (8 µm; 250 × 2 mm) and Reprosil Chiral AM (5 µm; 250 × 2 mm) were purchased from Dr. Maisch GmbH, Ammerbuch, Germany. Chiracel OB-H (250 × 2 mm) was from Daicel, and silicic acid (250 × 2 mm; 5 µm, Kromasil) from ChromTech. pPICZαA, Pichia pastoris (strain X-33), phleomycin (Zeocin), and yeast nitrogen base were from Invitrogen. Oligonucleotides for PCR were obtained from TIB Molbiol (Berlin, Germany). Recombinant 13R-MnLOX was expressed in P. pastoris (strain X-33) as a secreted protein containing 602 or 580 C-terminal amino acids (pPICZαA-MnLOX_602 and pPICZαA-MnLOX_580, respectively) and purified as described (17Su C. Oliw E.H. Manganese lipoxygenase. Purification and characterization.J. Biol. Chem. 1998; 273: 13072-13079Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 26Cristea 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, 35Wennman A. Jerneren F. Hamberg M. Oliw E.H. Catalytic convergence of Mn- and Fe-lipoxygenases by replacement of a single amino acid.J. Biol. Chem. 2012; 287: 31757-31765Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Chemically competent Escherichia coli (NEB5α) were from New England Biolabs. Restriction enzymes were from New England Biolabs and Fermentas. Plasmid Midi kits and Qiagen quick gel extraction kits were from Qiagen. M. salvinii (CBS 288.52 and CBS 254.34) were from Centraalbureau voor Schimmelcultures (Baarn, The Netherlands). Equipment and reagents for SDS-PAGE and real-time PCR (SYBR Green Supermix; iCycler iQ) were from Bio-Rad. Prestained protein ladder (Page Ruler) and colloidal Coomassie protein staining (Page-Blue) for SDS-PAGE were from Fermentas. M. salvinii was first grown on oatmeal or potato dextrose agar (1–2 weeks, 22°C) (20Krause R.A. Webster R.K. Morphology, taxonomy, and sexuality of rice stem rot fungus, Magnaporthe Salvinii (Leptosphaeria Salvinii).Mycologia. 1972; 64: 103-114Crossref Google Scholar), and then in liquid complete medium (per liter: 10 g glucose, 3 g NaNO3, 0.26 g KCl, 0.13 g MgSO4, 0.76 g KH2PO4, 2 g peptone, 1 g yeast extract, 1 g casamino acids, trace metals, and vitamin solution) under fluorescent light for (1–2 weeks, 22°C) with no or only slight agitation. The mycelia were separated by filtration, washed with saline, blotted dry, and ground to a fine powder with liquid nitrogen. This nitrogen powder was stored at −80°C and used for isolation of RNA and DNA and assay of oxylipin biosynthesis. For enzyme assay, the powder was homogenized in 0.1 M KHPO4 buffer (pH 7.4)/2 mM EDTA/0.04% Tween-20, centrifuged (13,000 g; 4°C, 10 min), and the supernatant was incubated with 100 µM 18:2n-6 or 18:3n-3 on ice for 30–40 min. The growth medium was concentrated 50–100 times by diafiltration (Amicon Ultracel-30K, Millipore), washed with five volumes 0.1 M NaBO3 buffer (pH 9.0), and stored at 4°C. A small aliquot (1–5 µl) of this concentrate was assayed for LOX activity with 100 µM 18:2n-6 or 18:3n-3 and for EAS activity with 40–100 µM 9S-HPODE (or other hydroperoxides) in 0.2–0.4 ml 0.1 M NaBO3 buffer (pH 9.0) for 10–30 min (22°C). Products were extracted on a cartridge of C18 silica (SepPak/C18; Waters). A large aliquot (200 µl) was subjected to gel filtration [0.05 M KHPO4 buffer (pH 7.4)/0.15 M NaCl/2 mM NaN3/0.04% Tween 20], and the fractions were assayed directly for LOX and EAS activity with 18:2n-6 and 9S-HPODE as substrates. Comparison of the deduced protein from the M. salvinii LOX mRNA (GenBank, AX590415) with 13R-MnLOX of G. graminis (GenBank, AAK81882) suggested an analogous N-terminal secretion peptide of 17 amino acids (SignalP 3.0 Server). The open reading frame without signal peptide was ordered from GenScript (Wheelock House; Hong-Kong) in pUC57, flanked by restriction sites for EcoRI and XbaI (1,815 bp). The open reading frame was ligated into the pPICZαA vector. This plasmid, pPICZαA_salvLOX, was then used to transform E. coli (NEB5α). We replaced Phe347 of 13R-MnLOX_580 with Leu, Val, and Ala by site-directed mutagenesis of pPICZαA_MnLOX_580 with Pfu DNA polymerase and primer pairs (forward and the reverse complement primer F347L: 5′-ggttctgggaccaaaacttaggcctgcccgcctcggccgcc; F347V, 5′-ggttctgggaccaaaacgttggcctgcccgcctcggccg; F347A, 5′-cggccgaggcgggcaggccagcgttttggtcccagaacc). In the same way, we replaced Leu350 of 9S-MnLOX with Phe and Met (L350F, 5′-gcgggttttgggaccagaactttggcctgcccgccacggcgg; L350M, 5′-gcgggttttgggaccagaacatgggcctgcccgccacggc). The PCR product was restricted with DpnI and transformed into E. coli (NEB5α). The Thr294 insert (ACT) in the His-Val-Leu-Phe-His pentamer was also ordered as a 492 bp sequence of the cDNA of 13R-MnLOX (GenScript) in pUC57. pUC57 with this insert was restricted with BspI and the fragment was used to replace the corresponding sequence in pPICZαA_MnLOX_580. The resulting pPICZαA_MnLOX_580_Tins was modified by replacements of Thr294 with Gly by site-directed mutagenesis using Pfu DNA polymerase and a primer pair (5′-gatgtaccacgtgctcttcggtcacacgatccccgagatcgtg-3′, and its complementary primer). All constructs were confirmed by sequencing at Uppsala Genome Center (Uppsala University). M. salvinii sporulates in growth media containing rice leaves sterilized by chemicals or by heat (20Krause R.A. Webster R.K. Morphology, taxonomy, and sexuality of rice stem rot fungus, Magnaporthe Salvinii (Leptosphaeria Salvinii).Mycologia. 1972; 64: 103-114Crossref Google Scholar). We therefore compared the expression of 9S-MnLOX mRNA by analysis of cDNA prepared from M. salvinii (CBS 288.54) grown in complete medium with or without autoclaved rice leaves (7 days, 24°C, slow agitation). Total RNA was prepared from nitrogen powder using the TRI reagent according to the manufacturer's protocol with the addition of a sonication step (30s ×10, maximal intensity) and treated with DNaseI (Promega). cDNA was synthesized with Superscript III (Invitrogen), and 250 ng was used as template for real-time PCR with SYBRGreen. cDNA was prepared from three different experiments and analyzed in triplicate. The two primers for 9S-MnLOX (forward, 5′-acagcgtatcgtgaagccaa; reverse, 5′-ttccaatggccgtcgtagag) were designed to span exon/exon boundaries to distinguish amplification of genomic DNA and cDNA by agarose gel electrophoresis. Relative expression levels were determined by correction for mRNA of the housekeeping gene β-tubulin (GenBank, AF396004.1; forward, 5′-cagcagatgttcgaccccaa; reverse, 5′-atctggtcctcgacctcctt). Melting curves and cycle threshold (CT) values were generated by the iCycler software (Bio-Rad) and corrected for β-tubulin expression, and PCR efficiency was calculated from serial dilutions of cDNA (iCycler software). Primer efficiency was determined to be ∼99%. Amplification data were analyzed with the comparative CT method (2−ΔΔCt) (36Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar). P. pastoris was transformed with pPICZαA expression plasmids as described (26Cristea 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). Transformants were selected on agar plates with yeast/peptone/dextrose/sorbitol/phleomycin (100 µg/ml), following instructions from Invitrogen. PCR of genomic DNA confirmed the insertion of the pPICZαA expression plasmids. The yeast colonies were first grown in buffered glycerol complex medium (BMGY) to generate biomass, washed, and grown in buffered minimal methanol (BMM) (26Cristea 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). Protein expression was induced by addition of 0.5% methanol daily. The recombinant proteins were secreted into the expression medium. The supernatant (0.25–0.5 l) was collected after 3–5 days for 9S-MnLOX and 5 days for 13R-MnLOX, by centrifugation (2,000 g, 5 min). The pH of the supernatant was adjusted from pH 3–4 to pH 7 with 10 M KOH and centrifuged, and the supernatant was assayed for LOX activity directly or after diafiltration (30 K, 2,000 g, 4°C). Recombinant 13R-MnLOX was purified as described (26Cristea 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: 201-211Cross
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