Fredrik Jernerén

Identification of Dioxygenases Required for Aspergillus Development STUDIES OF PRODUCTS, STEREOCHEMISTRY, AND THE REACTION MECHANISM

Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.

Manganese lipoxygenase oxidizes bis-allylic hydroperoxides and octadecenoic acids by different mechanisms

Manganese lipoxygenase (MnLOX) oxidizes (11R)-hydroperoxylinolenic acid (11R-HpOTrE) to a peroxyl radical. Our aim was to compare the enzymatic oxidation of 11R-HpOTrE and octadecenoic acids with LOO-H and allylic C-H bond dissociation enthalpies of ~88 and ~87kcal/mol. Mn(III)LOX oxidized (11Z)-, (12Z)-, and (13Z)-18:1 to hydroperoxides with R configuration, but this occurred at insignificant rates (<1%) compared to 11R-HpOTrE. We next examined whether transitional metals could mimic this oxidation. Ce(4+) and Mn(3+) transformed 11R-HpOTrE to hydroperoxides at C-9 and C-13 via oxidation to a peroxyl radical at C-11, whereas Fe(3+) was a poor catalyst. Our results suggest that MnLOX oxidizes bis-allylic hydroperoxides to peroxyl radicals in analogy with Ce(4+) and Mn(3+). The enzymatic oxidation likely occurs by proton-coupled electron transfer of the electron from the hydroperoxide anion to Mn(III) and H(+) to the catalytic base, Mn(III)OH(-). Hydroperoxides abolish the kinetic lag times of MnLOX and FeLOX by oxidation of their metal centers, but 11R-HpOTrE was isomerized by MnLOX to (13R)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid (13R-HpOTrE) with a kinetic lag time. This lag time could be explained by two competing transformations, dehydration of 11R-HpOTrE to 11-ketolinolenic acid and oxidation of 11R-HpOTrE to peroxyl radical; the reaction rate then increases as 13R-HpOTrE oxidizes MnLOX with subsequent formation of two epoxyalcohols. We conclude that oxidation of octadecenoic acids and bis-allylic hydroperoxides occurs by different mechanisms, which likely reflect the nature of the hydrogen bonds, steric factors, and the redox potential of the Mn(III) center.

Expression of 5,8-LDS of Aspergillus fumigatus and its dioxygenase domain : a comparison with 7,8-LDS, 10-dioxygenase, and cyclooxygenase

5,8-Linoleate diol synthase (5,8-LDS) of Aspergillus fumigatus was cloned, expressed, and compared with 7,8-LDS of the Take-all fungus. Replacements of Tyr and Cys in the conserved YRWH and FXXGPHXCLG sequences abolished 8R-dioxygenase (8-DOX) and hydroperoxide isomerase activities, respectively. The predicted α-helices of LDS were aligned with α-helices of cyclooxygenase-1 (COX-1) to identify the 8-DOX domains. N-terminal expression constructs of 5,8- and 7,8-LDS (674 of 1079, and 673 of 1165 residues), containing one additional α-helix compared to cyclooxygenase-1, yielded prominent 8R-DOX activities with apparently unchanged or slightly lower substrate affinities, respectively. Val-328 of 5,8-LDS did not influence the position of oxygenation in contrast to the homologous residues Val-349 of COX-1 and Leu-384 of 10R-dioxygenase. We conclude that ~675 amino acids are sufficient to support 8-DOX activity.

Gene Deletion of 7,8-Linoleate Diol Synthase of the Rice Blast Fungus STUDIES ON PATHOGENICITY, STEREOCHEMISTRY, AND OXYGENATION MECHANISMS

Linoleate diol synthases (LDS) are heme enzymes, which oxygenate 18:2n-6 sequentially to (8R)-hydroperoxylinoleic acid ((8R)-HPODE) and to (5S,8R)-dihydroxy-, (7S,8S)-dihydroxy-, or (8R,11S)-dihydroxylinoleic acids (DiHODE). The genome of the rice blast fungus, Magnaporthe oryzae, contains two genes with homology to LDS. M. oryzae oxidized 18:2n-6 to (8R)-HPODE and to (7S,8S)-DiHODE, (6S,8R)-DiHODE, and (8R,11S)-HODE. Small amounts of 10-hydroxy-(8E,12Z)-octadecadienoic acid and traces of 5,8-DiHODE were also detected by liquid chromatography-mass spectrometry. The contribution of the 7,8-LDS gene to M. oryzae pathogenicity was evaluated by replacement of the catalytic domain with hygromycin and green fluorescent protein variant (SGFP) cassettes. This genetically modified strain Δ7,8-LDS infected rice leaves and roots and formed appressoria and conidia as the native fungus. The Δ7,8-LDS mutant had lost the capacity to biosynthesize all the metabolites except small amounts of 8-hydroxylinoleic acid. Studies with stereospecifically deuterated linoleic acids showed that (8R)-HPODE was formed by abstraction of the pro-S hydrogen at C-8 and antarafacial oxygenation, whereas (7S,8S)-DiHODE and (8R,11S)-DiHODE were formed from (8R)-HPODE by suprafacial hydrogen abstraction and oxygenation at C-7 and C-11, respectively. A mac1 suppressor mutant (Δmac1 sum1–99) of M. oryzae, which shows cAMP-independent protein kinase A activity, oxygenated 18:2n-6 to increased amounts of (10R)-HPODE and (5S,8R)-DiHODE. Expression of the 7,8-LDS gene but not of the second homologue was detected in the suppressor mutant. This suggests that PKA-mediated signaling pathway regulates the dioxygenase and hydroperoxide isomerase activities of M. oryzae.

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

Background: Allene oxide synthases (AOS) of the CYP74 family are present in plants, but AOS of fungi have not been characterized. Results: Expression of dioxygenase-cytochrome P450 fusion proteins of Aspergillus terreus reveals a novel AOS. Conclusion: AOS of A. terreus forms compound II with catalytic similarities to CYP74 and CYP8A1. Significance: The fungal AOS protein sequence is unique with little homology to CYP74. Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn964 may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn878 in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln881 markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln881 with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1–4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus.

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity ( approximately 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain ( approximately 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of Aspergillusnidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with >65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus ☆

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-2H]18:2n-6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (alpha-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (gamma-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. alpha-Linolenic acid and 20:2n-6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but alpha- and gamma-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.

Epoxy alcohol synthase of the rice blast fungus represents a novel subfamily of dioxygenase-cytochrome P450 fusion enzymes

The genome of the rice blast fungus Magnaporthe oryzae codes for two proteins with N-terminal dioxygenase (DOX) and C-terminal cytochrome P450 (CYP) domains, respectively. One of them, MGG_13239, was confirmed as 7,8-linoleate diol synthase by prokaryotic expression. The other recombinant protein (MGG_10859) possessed prominent 10R-DOX and epoxy alcohol synthase (EAS) activities. This enzyme, 10R-DOX-EAS, transformed 18:2n-6 sequentially to 10(R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE) and to 12S(13R)-epoxy-10(R)-hydroxy-8(E)-octadecenoic acid as the end product. Oxygenation at C-10 occurred by retention of the pro-R hydrogen of C-8 of 18:2n-6, suggesting antarafacial hydrogen abstraction and oxygenation. Experiments with 18O2 and 16O2 gas confirmed that the epoxy alcohol was formed from 10R-HPODE, likely by heterolytic cleavage of the dioxygen bond with formation of P450 compound I, and subsequent intramolecular epoxidation of the 12(Z) double bond. Site-directed mutagenesis demonstrated that the cysteinyl heme ligand of the P450 domain was required for the EAS activity. Replacement of Asn965 with Val in the conserved AsnGlnXaaGln sequence revealed that Asn965 supported formation of the epoxy alcohol. 10R-DOX-EAS is the first member of a novel subfamily of DOX-CYP fusion proteins of devastating plant pathogens.

Catalytic Convergence of Manganese and Iron Lipoxygenases by Replacement of a Single Amino Acid

Background: 13R-MnLOX catalyzes suprafacial hydrogen abstraction and oxygenation in contrast to sLOX-1. Results: Mutation of one residue of 13R-MnLOX altered hydrogen abstraction and oxygenation to antarafacial. Conclusion: Replacement with the corresponding residue of soybean LOX-1 yielded catalytic convergence. Significance: The suprafacial oxygenation mechanism can be attributed to a single amino acid substitution. Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly332, Leu336, and Phe337) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (∼80–85, 15–20, and 2–3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe337 with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ∼100% R to 69–74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu336 with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly332 and Leu336 with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu336 can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe337 with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.

Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases

Seven Z-octadecenoic acids having the double bond located in positions 6Z to 13Z were photooxidized. The resulting hydroperoxy-E-octadecenoic acids [HpOME(E)] were resolved by chiral phase-HPLC-MS, and the absolute configurations of the enantiomers were determined by gas chromatographic analysis of diastereoisomeric derivatives. The MS/MS/MS spectra showed characteristic fragments, which were influenced by the distance between the hydroperoxide and carboxyl groups. These fatty acids were then investigated as substrates of cyclooxygenase-1 (COX-1), manganese lipoxygenase (MnLOX), and the (8R)-dioxygenase (8R-DOX) activities of two linoleate diol synthases (LDS) and 10R-DOX. COX-1 and MnLOX abstracted hydrogen at C-11 of (12Z)-18:1 and C-12 of (13Z)-18:1. (11Z)-18:1 was subject to hydrogen abstraction at C-10 by MnLOX and at both allylic positions by COX-1. Both allylic hydrogens of (8Z)-18:1 were also abstracted by 8R-DOX activities of LDS and 10R-DOX, but only the allylic hydrogens close to the carboxyl groups of (11Z)-18:1 and (12Z)-18:1. 8R-DOX also oxidized monoenoic C14-C20 fatty acids with double bonds at the (9Z) position, suggesting that the length of the omega end has little influence on positioning for oxygenation. We conclude that COX-1 and MnLOX can readily abstract allylic hydrogens of octadecenoic fatty acids from C-10 to C-12 and 8R-DOX from C-7 and C-12.