Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

Abstract

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-2H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the 2H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser348 in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser348, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs. The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-2H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the 2H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser348 in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser348, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs. Lipoxygenases (LOXs) belong to a common gene family (1.Kuhn H. Banthiya S. van Leyen K. Mammalian lipoxygenases and their biological relevance.Biochim. Biophys. Acta. 2015; 1851: 308-330Crossref PubMed Scopus (364) Google Scholar, 2.Horn T. Adel S. Schumann R. Sur S. Kakularam K.R. Polamarasetty A. Redanna P. Kuhn H. Heydeck D. Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling.Prog. Lipid Res. 2015; 57: 13-39Crossref PubMed Scopus (68) Google Scholar). The catalytic domain is formed by α-helices, which harbor five metal ligands. The 3D structure is known for over 10 enzymes from plants, animals, and bacteria with 13S-LOX of soybean LOX-1 (sLOX-1) and 8R-LOX of Plexaura homomalla as old and new prototypes (3.Minor W. Steczko J. Stec B. Otwinowski Z. Bolin J.T. Walter R. Axelrod B. Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution.Biochemistry. 1996; 35: 10687-10701Crossref PubMed Scopus (394) Google Scholar, 4.Neau D.B. Gilbert N.C. Bartlett S.G. Boeglin W. Brash A.R. Newcomer M.E. The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity.Biochemistry. 2009; 48: 7906-7915Crossref PubMed Scopus (85) Google Scholar, 5.Neau D.B. Bender G. Boeglin W.E. Bartlett S.G. Brash A.R. Newcomer M.E. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.J. Biol. Chem. 2014; 289: 31905-31913Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The latter was recently crystallized with its substrate in the active site (5.Neau D.B. Bender G. Boeglin W.E. Bartlett S.G. Brash A.R. Newcomer M.E. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.J. Biol. Chem. 2014; 289: 31905-31913Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Information on the active site and reaction mechanism has also been gained by site-directed mutagenesis and by analysis of various substrates, deuterated fatty acids, and LOX inhibitors (1.Kuhn H. Banthiya S. van Leyen K. Mammalian lipoxygenases and their biological relevance.Biochim. Biophys. Acta. 2015; 1851: 308-330Crossref PubMed Scopus (364) Google Scholar, 6.Haeggström J.Z. Funk C.D. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease.Chem. Rev. 2011; 111: 5866-5898Crossref PubMed Scopus (599) Google Scholar). With few exceptions, LOXs oxidize polyunsaturated fatty acids with one or more cis,cis-1,4-pentadiene units to hydroperoxides, which in many cases are precursors of biological mediators (1.Kuhn H. Banthiya S. van Leyen K. Mammalian lipoxygenases and their biological relevance.Biochim. Biophys. Acta. 2015; 1851: 308-330Crossref PubMed Scopus (364) Google Scholar, 6.Haeggström J.Z. Funk C.D. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease.Chem. Rev. 2011; 111: 5866-5898Crossref PubMed Scopus (599) Google Scholar, 7.Andreou A. Brodhun F. Feussner I. Biosynthesis of oxylipins in non-mammals.Prog. Lipid Res. 2009; 48: 148-170Crossref PubMed Scopus (236) Google Scholar). Mammalian LOXs initiate biosynthesis of leukotrienes and epoxy alcohols, which are involved in allergic inflammation, asthma, formation of the skin-water barrier, and cancer development (6.Haeggström J.Z. Funk C.D. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease.Chem. Rev. 2011; 111: 5866-5898Crossref PubMed Scopus (599) Google Scholar, 8.Zheng Y. Yin H. Boeglin W.E. Elias P.M. Crumrine D. Beier D.R. Brash A.R. Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope.J. Biol. Chem. 2011; 286: 24046-24056Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Plant LOXs form jasmonates and related oxylipins with functions in defense against pathogens and in wound healing (9.Wasternack C. Kombrink E. Jasmonates: structural requirements for lipid-derived signals active in plant stress responses and development.ACS Chem. Biol. 2010; 5: 63-77Crossref PubMed Scopus (202) Google Scholar, 10.Vicente J. Cascon T. Vicedo B. Garcia-Agustin P. Hamberg M. Castresana C. Role of 9-lipoxygenase and alpha-dioxygenase oxylipin pathways as modulators of local and systemic defense.Mol. Plant. 2012; 5: 914-928Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The N-terminal parts of mammalian and plant LOXs usually consist of β-sheets with homology to polycystin-1/LOX/α-toxin (PLAT) domains with regulatory functions. The N-terminal domains of fungal LOXs lack this homology, but they often contain a secretion signal (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar, 12.Heshof R. Jylha S. Haarmann T. Jorgensen A.L. Dalsgaard T.K. de Graaff L.H. A novel class of fungal 2014; PubMed Scopus Google Scholar). pathogens LOXs to and of LOXs also biosynthesis of and including biosynthesis of jasmonates (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar, in in the 2012; PubMed Scopus Google Scholar, F. Feussner I. in J. 2011; PubMed Scopus Google Scholar, H. C. and related from Fusarium Scopus Google Scholar, S. D. of Chem. C. Google Scholar, K. K. K. Biosynthesis of acid in a plant 2010; PubMed Scopus Google Scholar). Fusarium oxysporum was to acid over but the mechanism is H. C. and related from Fusarium Scopus Google Scholar). has in the of oxygenation of fatty acids by this The genome codes for of and a related and LOXs and F. oxysporum manganese LOX I. of a linoleate and an in a of Fusarium Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, I. F. of the a novel of Lipid Res. 2014; Full Text Full Text PDF PubMed Scopus Google form jasmonates from the of which is by plant and by to the of jasmonates A.R. aspects of and related 2009; PubMed Scopus Google Scholar). 13S-LOX and the for jasmonate biosynthesis (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar). Fo-MnLOX also but is known this other than its homology to the (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar). The of the family is 13R-MnLOX of the of Gaeumannomyces C. of in a lipoxygenase and of local PubMed Scopus Google Scholar, C. M. of manganese lipoxygenase with a catalytic Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. E. of a as product and in a lipoxygenase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are of the A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google of the and of and and (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar, 12.Heshof R. Jylha S. Haarmann T. Jorgensen A.L. Dalsgaard T.K. de Graaff L.H. A novel class of fungal 2014; PubMed Scopus Google Scholar, C. M. of manganese lipoxygenase with a catalytic Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google LOXs with catalytic of F. oxysporum and the recently of the Y. S. T. T. of a lipoxygenase from the 2013; PubMed Scopus Google form a with to plant and LOXs than to MnLOXs (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar, 12.Heshof R. Jylha S. Haarmann T. Jorgensen A.L. Dalsgaard T.K. de Graaff L.H. A novel class of fungal 2014; PubMed Scopus Google Scholar). 13R-MnLOX was recently crystallized A. S. and a analysis of Biol. 2014; PubMed Scopus Google but the 3D structure has not been Electron paramagnetic resonance analysis suggests that and are in and MnLOXs in a in with other enzymes C. of in a lipoxygenase and of local PubMed Scopus Google Scholar, lipoxygenase to structure by paramagnetic Chem. Res. 2014; PubMed Scopus Google Scholar). The and in and A. S. of the of and Biophys. 2014; PubMed Scopus Google Scholar, M. Hamberg M. Biosynthesis and of by and PubMed Scopus Google Scholar). is by the acid to oxidation and of the metal A. S. of the of and Biophys. 2014; PubMed Scopus Google Scholar). F. oxysporum and Colletotrichum belong to the fungal pathogens of biology, and they the of a of and R. A. M. R. J. 10 fungal pathogens in plant Plant 2012; PubMed Scopus Google Scholar, K. F. K. Fusarium Rev. 2013; PubMed Scopus Google Scholar). MnLOXs are and to the by oxidation of plant and biosynthesis of and they are related to and which more than 13R-MnLOX for and and from or and from was The and from [11S-2H]18:2n-6 was as M. of hydrogen oxygenation of acid by and of 2011; PubMed Scopus Google Scholar). acids in and in at acid (HPODE), acid and by biosynthesis and by or by acid and by oxidation T. and analysis of fatty acids by PubMed Scopus Google Scholar). with and from was from and acid from and from for from from enzymes from and was from was from and from The proposed of the MnLOXs from F. and C. on one and predicted the of and and the of and secretion signals of predicted with the of the The of the proposed of C. was this was from the so that the and Cg-MnLOX and The of and their secretion signals from in the in with the secretion signal of The in E. by and with and P. was with as A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, M. C. of manganese lipoxygenase in and site-directed mutagenesis of metal Biophys. PubMed Scopus Google Scholar). on with at M. C. of manganese lipoxygenase in and site-directed mutagenesis of metal Biophys. PubMed Scopus Google Scholar). as at for on was by of Ser348 with a Phe and with an with the of site-directed mutagenesis The products with by and to E. P. was as and and of the in P. in in at was with to at at for in was by of for The was in the and The activity at the to of and the was The by was to the and was to with 10 The which by by and the which contained of the LOX was at the was and on a with on an or a The was with the and with and the for LOX activity by A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). The active the was by and the was by at or An was for or Fo-MnLOX was also and by with at at and with and as A. S. and a analysis of Biol. 2014; PubMed Scopus Google Scholar). acids with the and Cg-MnLOX in The products as LOX activity was on a The was with 18:2n-6 or in and the was at and isotope deuterium by of the enzymes with [11S-2H]18:2n-6 and and by the in at The reaction was from the of the The of Fo-MnLOX was by the reaction with 18:2n-6 from the of biosynthesis of and We the biosynthesis of which was from the of and by of the and analysis with of the signal of the of and and to with linoleate The was with the for We of Fo-MnLOX by Ser348Phe and site-directed mutagenesis as A. F. Hamberg M. of manganese and iron lipoxygenases by of a Biol. Chem. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). The by and and expressed and by as by on and with analysis was with a on an with a which at with acid for fatty The was to in a The was at the usually at for and for and the at was for which the was with a usually with at of fatty acids, a with at T. and analysis of fatty acids by PubMed Scopus Google Scholar). fatty acids on at with or on at with The of with and and by the on a an in a with an The was an and as was the analysis of Fo-MnLOX in was and of the with in the was by The was in with and by with and which a with to The was and as in the The was to with metal and the was to by at The and effect on catalytic activity was by of the with and 10 and with and 10 The reaction was with as The by of the with of 18:2n-6 in was for was for of Fo-MnLOX with 8R-LOX as a M. S. A. K. G. T. F. M. and structure Res. 2014; PubMed Scopus Google Scholar). was for the and expressed in and to the to the enzymes with at The enzymes by as in the and in of of the activity and an in specific activity from to by and in and a specific activity of analysis of Fo-MnLOX the and is in The of Fo-MnLOX was and Cg-MnLOX was The predicted also and by P. to and mainly with of secretion in and in 2012; PubMed Scopus Google Scholar). analysis showed a and that to by with and H. and oxidized 18:2n-6 to and The was oxidized by and to as the product with and Fo-MnLOX also formed of also with and the of the in analysis showed that Cg-MnLOX oxidized 18:2n-6 to and 13R-HPODE and whereas Fo-MnLOX formed and 13R-HPODE in a and was with in to of of by Fo-MnLOX with the 13R-MnLOX to 13R-MnLOX to 13R-HPODE and to M. Hamberg M. Biosynthesis and of by and PubMed Scopus Google Scholar). We that Fo-MnLOX the of is in to MnLOXs that mainly formed the We that Cg-MnLOX formed which was by 13R-MnLOX to not to was as which is the same formed by 13R-MnLOX and to is that the or at C-11 of 18:2n-6 and by is by the which results in and at the position of and the hydrogen is in the same analysis and of a of and which are formed by and is in of [11S-2H]18:2n-6 Cg-MnLOX and Fo-MnLOX. The reaction by the involved loss of the deuterium accompanied by a kinetic isotope effect not whereas the Fo-MnLOX-catalyzed reaction with retention of the and a isotope effect is of deuterium kinetic isotope of hydrogen in soybean lipoxygenase of and isotope PubMed Scopus Google Scholar). The analysis of the of the formed by Fo-MnLOX details the retention of the deuterium which was by the of The latter retention of the except for and We that produced by Fo-MnLOX, is formed by suprafacial hydrogen and MnLOXs by suprafacial hydrogen and M. C. E. of a as product and in a lipoxygenase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of products formed from 18:2n-6 by analysis at substrate is in an of 10 The of to with of and for the of biosynthesis of the was We fatty acids for oxygenation by Fo-MnLOX by analysis or 18:2n-6 was oxidized with The of oxidation of and and analysis showed that Fo-MnLOX oxidized at and The oxidation of and that the was not for of the in the active site of Fo-MnLOX. of the or oxidation of and The which as a was oxidized to and at a compared with and Fo-MnLOX and Cg-MnLOX oxidized soybean as from a rapid and in at as for Fo-MnLOX in with 18:2n-6 contained and of other fatty acids and as from and analysis of We compared the of oxidation of with that of The was oxidized at a than 18:2n-6 We that and 18:2n-6 are oxidized at was a substrate suggest that fatty acids to the active site of and Cg-MnLOX. and of Fo-MnLOX by to and The was We that the catalytic metal of Fo-MnLOX is and not The in the compared with the was of to the in a of analysis of Fo-MnLOX in showed a but with to the of a and of and is in with the of by of 13R-MnLOX C. M. of manganese lipoxygenase with a catalytic Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the catalytic metal of Fo-MnLOX is Mn. The of was as the was to by by as in the and of the with or 10 did not the catalytic with or 10 the oxidation of is that and the in the and the catalytic metal from oxidized to its active on the product formed from 18:2n-6 in to Fo-MnLOX. the product and formed in a of and and in a of mainly of the The of and The of oxidation of 18:2n-6 by this mutant on the was with oxidized 18:2n-6 and of as from and the of oxidation of the latter to by suggests that the active site of the mutant with the the same as was in the Fo-MnLOX. We a model of Fo-MnLOX with 8R-LOX as The the position of Ser348 and the Phe as as and to for of the substrate in the active is not We as that the homolog of F. oxysporum, Fo-MnLOX, the same oxidation mechanism as and but formed the of as a major the Cg-MnLOX homolog formed the of in with 13R-MnLOX. Fo-MnLOX by suprafacial hydrogen and whereas 9S-, and 13R-MnLOX form by the same Fo-MnLOX binds 18:2n-6 so that the proR hydrogen at C-11 is to the metal center, whereas the proS hydrogen is in 9S-, and is also formed by the iron LOX of the but this by hydrogen and oxygenation A. C. Hamberg M. Feussner I. A from that has an iron as Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). of MnLOXs in with to the suprafacial hydrogen and oxygenation mechanism M. C. E. of a as product and in a lipoxygenase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). are not to β-fragmentation by and or by at in with 9S-, and M. Hamberg M. Biosynthesis and of by and PubMed Scopus Google Scholar, the of by manganese Lipid Res. Full Text Full Text PDF PubMed Scopus Google is the of the is for β-fragmentation A. C. Hamberg M. Feussner I. A from that has an iron as Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). A catalytic with but and Cg-MnLOX that also The of and with acid and oxidize 18:2n-6 to with and also oxidize in with 13R-MnLOX the of by manganese Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). in with a common substrate which binds the of fatty acids and with the at the The 3D structure of 8R-LOX with arachidonic was recently (5.Neau D.B. Bender G. Boeglin W.E. Bartlett S.G. Brash A.R. Newcomer M.E. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.J. Biol. Chem. 2014; 289: 31905-31913Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The active site is and mainly by acid the homology the active of and for the active site of 8R-LOX (5.Neau D.B. Bender G. Boeglin W.E. Bartlett S.G. Brash A.R. Newcomer M.E. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.J. Biol. Chem. 2014; 289: 31905-31913Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, M.E. Brash A.R. The structural for in lipoxygenase 2015; PubMed Scopus Google Scholar). The MnLOXs and Fo-MnLOX are in Fo-MnLOX with Ser348 and of Phe and in the position in the other MnLOXs. also that the of the metal not the as from their The and of the with whereas A. and with of acid of the arachidonic acid site of 8R-LOX and acids of and to of G. from of M. in one of the of 8R-LOX not with arachidonic acid of 8R-LOX not with arachidonic acid of 13R-MnLOX is for the suprafacial hydrogen and in five or all are in The that are by an An acid in the is 13R-MnLOX of G. from of M. in one of the The of 8R-LOX not with arachidonic acid (5.Neau D.B. Bender G. Boeglin W.E. Bartlett S.G. Brash A.R. Newcomer M.E. Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase.J. Biol. Chem. 2014; 289: 31905-31913Abstract Full Text Full Text PDF PubMed Scopus (49) Google of 13R-MnLOX is for the suprafacial hydrogen and A. F. Hamberg M. of manganese and iron lipoxygenases by of a Biol. Chem. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). in a new in five or all are in The that are by an An acid in the is We of and Ser348 of Fo-MnLOX with the and Phe in the other MnLOXs the product oxidized 18:2n-6 to products in with the on the of the products and formed in a of and the of from mainly to mainly and the of from to S. has that of a acid in the active site of LOXs the product (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar, R. C. J. P. P. Kuhn H. M. of the for the of mammalian Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus (68) Google Scholar, G. Brash A.R. A active site oxygenation in is to the position of PubMed Scopus Google Scholar). Ser348 of Fo-MnLOX with of 8R-LOX and this is the of the arachidonic acid site of which is as the has been to the position of the substrate in the active site of and the A. of novel manganese and epoxy by the Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, A. F. Hamberg M. of manganese and iron lipoxygenases by of a Biol. Chem. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, R. E. A for lipoxygenase PubMed Scopus Google Scholar). Our that at this the hydrogen at C-11 from to which is a of an and from acid by a Biophys. Acta. PubMed Scopus Google Scholar). The that Fo-MnLOX and not support an the of 18:2n-6 by the Phe so that a new with the proS hydrogen at C-11 to the catalytic metal Our results a by which the the of a specific A and of was the in of from to are to for their and in their active are to the of known to in this that the of Fo-MnLOX at the Ser348 to was to that are from F. oxysporum and are to Fo-MnLOX. and metal analysis that was in large over analysis and showed that was mainly and metal analysis also that was at high in of with by The signal of was not by to the same as the signal of to the of metal to binds more than and D. and Biol. Chem. 2014; 289: Full Text Full Text PDF PubMed Scopus Google Scholar). The high in the to or the active the latter a catalytic role of in is and is of to Fo-MnLOX and of F. enzymes but (11.Brodhun F. Cristobal-Sarramian A. Zabel S. Newie J. Hamberg M. Feussner I. An iron 13S-lipoxygenase with an alpha-linolenic acid specific hydroperoxidase activity from Fusarium oxysporum.PLoS One. 2013; 8: e64919Crossref PubMed Scopus (58) Google Scholar). Fo-MnLOX oxidized all of and and formed with stereoselectivity. The oxidation of by to as the or is by the which the or of the to as as is the LOX for fungal biosynthesis of jasmonates from in the plant Fo-MnLOX, and the in the of F. oxysporum that lack on the and biological of a of oxylipins The secretion signals of fungal and suggest an in MnLOXs are expressed by fungal which the plant in with G. M. and M. LOXs to of in and (1.Kuhn H. Banthiya S. van Leyen K. Mammalian lipoxygenases and their biological relevance.Biochim. Biophys. Acta. 2015; 1851: 308-330Crossref PubMed Scopus (364) Google Scholar). LOXs a on plant and from 13R-MnLOX in Fo-MnLOX by suprafacial hydrogen and 11-hydroperoxides of 18:2n-6 and to rapid β-fragmentation by but not by and Cg-MnLOX. the of the of and are also in and and but are by in and The reaction mechanism of Fo-MnLOX by of Phe with Ser348