Florian Brodhun

Stefan Schneider

Cornelia Göbel

Ellen Hornung

Ivo Feussner

PpoC from Aspergillus nidulans is a Fusion Protein with only one active Heme

Keywords: Fatty acid dioxygenase, oxylipin formation, psi factor forming oxygenase

In Aspergillus nidulans psi factor producing oxygenases (Ppos) are required for the production of so-called psi factors (precocious sexual inducer) -compounds that control balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different heme containing domains are predicted: a fatty acid heme peroxidase/dioxygenase domain for the N-terminal region and a P450 heme thiolate domain for the C-terminal region. While PpoA was shown to use both heme domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8hydroperoxide isomerisation), we found that PpoC apparently only harbours a functional heme peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyzes mainly the dioxygenation of linoleic acid (18:2 Δ9Z,12Z ), yielding 10-hydroperoxy linoleic acid (10-HPODE). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-keto linoleic acid (10-KODE) and 10-hydroxy linoleic acid (10-HODE). In parallel, decomposition of 10-HPODE into 10-oxo decenoic acid (10-ODA) and volatile C 8 alcohols that are inter alia responsible for the characteristic mushroom flavour. Beside these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE to the respective epoxy alcohols: 12,13-Epoxy-8-hydroxy octadecenoic acid and 12,13-epoxy-10hydroxy octadecenoic acid. By using site directed mutagenesis we could demonstrate that both enzymes share a similar mechanism for the oxidation of 18:2 Δ9Z,12Z : They both use a conserved tyrosine for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain.

INTRODUCTION

In Aspergillus nidulans (teleomorph Emericella nidulans) oxidized unsaturated fatty acids, so-called oxylipins, derived from endogenous fatty acids, have been described to regulate the developmental life cycle of the fungus [START_REF] Mazur | Structural elucidation of sporogenic fatty acid metabolites from Aspergillus nidulans[END_REF][START_REF] Mazur | Structure and synthesis of sporogenic Psi factors from Aspergillus nidulans[END_REF][START_REF] Champe | Isolation of a sexual sporulation hormone from Aspergillus nidulans[END_REF][START_REF] Champe | An endogenous inducer of sexual development in Aspergillus nidulans[END_REF][START_REF] Calvo | Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans[END_REF][START_REF] Calvo | Relationship between secondary metabolism and fungal development[END_REF][START_REF] Tsitsigiannis | Oxylipins as developmental and host-fungal communication signals[END_REF]. In A. nidulans these oxylipins are collectively called psi factors (precocious sexual inducer). Psi factors are a mixture of oxidized 18:1 Δ9Z , 18:2 Δ9Z,12Z and 18:3 Δ9Z,12Z,15Z , where x:y Δz is a fatty acid containing x carbons and y double bonds in position z counting from the carboxyl group [START_REF] Andreou | Biosynthesis of oxylipins in non-mammals[END_REF] and are termed psiβ, psiα and psiγ, respectively. The number and the position of the hydroxyl groups on the fatty acid backbone leads to further classification of psi factors: psiB (OH at C-8, e.g. (8R)-HODE 2 ), psiA (OH at C-5 and C-8, e.g. (5S,8R)-DiHODE) and psiC (OH at C-8 and δ-lactone ring) [START_REF] Mazur | Structural elucidation of sporogenic fatty acid metabolites from Aspergillus nidulans[END_REF][START_REF] Mazur | Structure and synthesis of sporogenic Psi factors from Aspergillus nidulans[END_REF]. The genes encoding enzymes that are involved in the production of psi factors have been first identified by Keller and co-workers and were termed psi factor producing oxygenases (Ppos). The genome of A. nidulans harbours three different genes possibly encoding Ppos: ppoA, ppoB and ppoC [START_REF] Tsitsigiannis | The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans[END_REF][START_REF] Tsitsigiannis | Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence[END_REF][START_REF] Tsitsigiannis | Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans[END_REF]. The same group could show that deletion of these genes had a significant effect (i) on the developmental ratio between asexual conidiospores and sexual ascospores, (ii) on the production of psi factors and (iii) on the production of secondary metabolites, the mycotoxins [START_REF] Tsitsigiannis | The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans[END_REF][START_REF] Tsitsigiannis | Three putative oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus nidulans[END_REF][START_REF] Tsitsigiannis | Oxylipins act as determinants of natural product biosynthesis and seed colonization in Aspergillus nidulans[END_REF][START_REF] Tsitsigiannis | Endogenous lipogenic regulators of spore balance in Aspergillus nidulans[END_REF]. Ppo enzymes show a significant sequence identity to mammalian prostaglandin endoperoxide H synthase (25-29 % identity) and fungal 7,8-linoleate diol synthase (40-45 % identity) [START_REF] Tsitsigiannis | Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin production and virulence[END_REF]. In contrast to 7,8-linoleate diol synthase a search for conserved domains using the National Centre for Biotechnology Information (NCBI) database predicted for Ppo enzymes the presence of two different heme domains: a heme Expression of recombinant Ppo-enzymes in E. coli E.coli BL21* cells containing pET24-PpoC and the plasmid pGro7 (encoding the Chaperone groES-groEL) (Takara Bio Inc. Otsu, Japan) were cultivated in 2xYT broth containing 50 µg/mL chloramphenicol, 25 µg/mL kanamycin, 25 µg/mL arabinose to induce the expression of the chaperone and 0.02 mM δ-aminolaevulinic acid. Cells were grown to an OD 600 of 0.6-0.8. Induction of the expression of the recombinant protein was performed by adding isopropyl-beta-Dthiogalactopyranoside to a final concentration of 0.1 mM and cultures were cultivated for 3 d shaking at 16° C. Cells were harvested by centrifugation at 4,000 x g and 6° C for 10 min. The pellet was shock frozen in liquid nitrogen and stored at -20° C.

Cell lysis and protein purification

Harvested cells of 500 mL culture expressing recombinant PpoC were resuspended in 50 ml 50 mM Tris/HCl, pH 8.0. After addition of lysozyme to a final concentration of 0.1 mg/mL and phenylmethylsulfonylfluorid to a final concentration of 0.2 mM, cells were incubated for 30 min on ice. Cells were sonified and this extract was centrifuged at 54,000 x g and 4° C for 20 min to clear debris, yielding crude bacterial extract. The supernatant obtained from cell lysis was loaded on a Source 30Q resin (25 ml, XK 16/20 column; GE Healthcare, Munich, Germany) at a flow rate of 1 mL/min using an ÄKTA-Prime-System (GE Healthcare, Munich, Germany). The column was washed with 50 ml of 50 mM Tris/HCl (pH 8.0) and protein was eluted with a linear gradient of 0-0.3 M NaCl in 50 mM Tris/HCl (pH 8.0) within 30 min at a flow rate of 2 ml/min. Fractions of 2 ml were collected. Fractions with highest activity (as judged by Clark-type O 2 -electrode) and highest purity (as judged by SDS-PAGE) were combined and concentrated to a final volume of 2 ml using Vivaspin 20 (100.000 MWCO) (Sartorius Stedim Biotech GmbH, Goettingen, Germany). For gel filtration the concentrated protein extract was loaded on a Superdex S200 26/60 pg column (GE Healthcare, Munich, Germany) equilibrated with 50 mM Tris/HCl (pH 8.0) containing 1 M NaCl. Elution was performed at a flow rate of 2 ml/min. Fractions containing enzyme activity were combined and loaded on a desalting 26/10 column (GE Healthcare, Munich, Germany) equilibrated with 50 mM Tris/HCl (pH 8.0). The desalted protein solution was loaded on a MonoQ 10/10-column (GE Healthcare, Munich, Germany). After loading and washing with 50 mM Tris/HCl (pH 8.0) the protein was eluted at 3 ml/min with a linear gradient to 50 mM Tris/HCl (pH 8.0) containing 1 M NaCl within 30 min. Recombinant PpoA and related mutants (PpoA_V328A & PpoA_V328L) were purified as described [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF].

Sodium salts of 18:1 Δ9Z , 18:2 Δ9Z,12Z , and 18:3 Δ9Z,12Z,15Z . Kinetic parameters were determined by fitting seven data points to Michaelis-Menten equations by using the software Origin 6.0.

Determination of the pH-optimum

The pH-optimum was determined by measuring initial rates of oxygen consumption at a defined pHvalue using a Clark-type O 2 -electrode (Rank Brothers, Bottisham, Cambridge, UK): 200 µL PpoC bacterial crude extract were added to 800 µL of buffer containing 100 µM of the sodium salt of 18:2 Δ9Z,12Z . The following buffers were used for the different pH-ranges: 0.2 M acetate buffer (pH 4.5-5.5), 0.2 M phosphate buffer (pH 5.5-8.0), 0.2 M borate buffer (pH 8.0-10.5), respectively.

RESULTS

PpoC is a fusion protein of the CYP6001 family

BLAST search analysis of PpoC predicts the presence of two domains in its sequence: Within the Nterminal region of the protein a heme peroxidase/dioxygenase domain is predicted whereas a P450 heme thiolate domain is proposed for the C-terminal part. Therefore, PpoC from A. nidulans was assigned as CYP6001C1 by Dr. D. R. Nelson of the P450 Nomenclature Committee (Department of Molecular Sciences, University of Tennessee). A similar domain architecture has also been described recently for PpoA, another member of the Ppo-family of A. nidulans, for which functionality of both domains has been proven [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. Sequence alignments of the predicted N-terminal heme peroxidase/dioxygenase domain of PpoC with those of A. fumigatus PpoC (Fig. 2A, AfPpoC), A. nidulans PpoA (Fig. 2A, AnPpoA), G. graminis 7,8-linoleate diol synthase (Fig. 2A, GgLDS), sheep prostaglandin endoperoxide H synthase-1 (Fig. 2A, sheep PGHS1) and sheep prostaglandin endoperoxide H synthase-2 (Fig. 2A, sheep PGHS2) showed that residues reported to be essential for the catalytic dioxygenase activity are conserved in all six sequences. However, sequence alignments of the predicted C-terminal P450 domain of PpoC with those of known P450 heme thiolate enzymes, the recently described A. nidulans PpoA [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF] and A. fumigatus PpoC [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF], showed that the crucial cysteine, which is known as the 5 th heme iron ligand, is neither conserved in PpoC from A. nidulans nor from A. fumigatus. Instead of a cysteine a glycine and a phenylalanine, respectively, are found at the particular position of the PpoC sequences (Fig. 2B). This may indicate that the P450 domain is not functional and does not show catalytic activity (vide infra).

Cloning and functional expression of PpoC

In order to analyze the enzymatic reaction catalyzed by PpoC from A. nidulans, its cDNA was cloned from vegetative and sexual stages of A. nidulans by using specific primers. Sequencing of the resulting plasmid confirmed the ORF with 3933 bp and 1117 amino acids, respectively, showing one amino acid residue exchange (S215P) according to the published genome sequence [START_REF] Galagan | Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae[END_REF]. Functional expression of PpoC was performed in E. coli Bl21* cells and optimal cultivation conditions (16 °C, 3 d) were determined. Expression of PpoC could be enhanced by co-expression of Chaperone groES-groEL and the addition of 0.2 mM δ-aminolaevulinic acid before induction (heme biosynthesis). Protein expression was verified by SDS-PAGE (Fig. 3A).

Purification of PpoC

In order to purify PpoC a protocol was used that based on the previously described protocol for PpoA purification [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. Extraction and solubilization of PpoC using crude extract from bacteria was performed with 50 mM Tris/HCl, pH 8.0. The addition of detergents and NaCl to the extraction buffer did not improve protein extraction. For the initial purification step a Source 30Q resin was chosen to isolate and concentrate PpoC from the crude cell extract. Fractions showing the highest activity were combined and further purified using size exclusion chromatography. The native molecular mass of PpoC using gel-filtration chromatography was 480 kDa. This was 4-fold larger than that determined by SDS-PAGE. Therefore, the native protein is a homo tetramer as it has previously been demonstrated for 7,8-linoleate diol synthase [START_REF] Su | Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein[END_REF] and PpoA [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. With the final chromatography step using a MonoQ column most of the remaining contaminants were removed. After each purification step the protein fractions showing the highest activity were combined and subjected to SDS-PAGE (Fig. 3A). A protein band at approximately 120 kDa became subsequently more prominent. Using this protocol we were able to purify approx. 0.5-2 mg of protein from 500 ml of cell culture. The activity A c c e p t e d M a n u s c r i p t of the pure protein was estimated to be 6.5 µmol [O 2 ]/(min*mg) using the Clark-type O 2 -electrode. However, we found that the activity of the purified protein decreased significantly within a few hours. The addition of neither 10 % glycerol nor 5 mM EDTA to the buffer improved PpoC stability.

Spectral analysis and prosthetic groups

The UV/vis absorption spectrum of purified PpoC is shown in Fig. 3B. The highest absorption maxima were at 280 nm (probably due to aromatic amino acid site chains, not shown) and 403 nm (γ, Soret band) indicating the presence of heme. Additionally two weaker maxima at approximately 530 nm and 630 nm could be detected and are indicated by arrows. However, the heme absorption spectrum appeared to be weak -a fact that might indicate that heme was partially lost during the purification procedure.

Identification of products formed by recombinant PpoC

As we found purified PpoC to be highly unstable under our assay conditions, we decided to investigate most of the biochemical properties of PpoC by using bacterial crude extract of PpoC expressing E. coli cells and cells expressing the empty vector as controls. Bacterial crude extract of PpoC expressing cells was incubated with 18:2 Δ9Z,12Z for 30 min on a shaker. After extraction of fatty acids and their derivatives we analyzed product formation with RP-HPLC/MS 2 . We detected the formation of 10-HPODE (m/z 311, RT 22.1 min), 8/10-HODE (m/z 295, RT 19.5 min/20.9 min), 10-KODE (m/z 293, RT 22.6 min) and 10-ODA (m/z 183, RT 3.8 min) (Fig. 4A-D). Additionally we found the formation of 12,13-epoxy-10-hydroxy octadecenoic acid (m/z 311, RT 9.9 min; Fig. 4A). The corresponding MS/MS spectra of 12,13-epoxy-10-hydroxy octadecenoic acid, 8-HODE, 10-HODE, 10-KODE and 10-ODA are shown in Supplemental Fig. 1A-E and are in agreement with previously reported spectra [START_REF] Garscha | Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism[END_REF][START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. Bacterial crude extract of empty vector controls did not metabolize 18:2 Δ9Z,12Z to any of these products. Comparing the relative amounts of different products formed during the incubation, we found that 10-H(P)ODE appeared to be the main products that are formed from 18:2 Δ9Z,12Z by PpoC (Fig 4E ), while 12,13-epoxy-10-hydroxy octadecenoic acid and 10-ODA accumulated to lesser extends. We next examined which products are formed by PpoC from other fatty acid substrates. We were particularly interested in products formed from fatty acids that occur endogenously in A. nidulans, such as other unsaturated C18-fatty acids. As supposed, 18:1 Δ9Z and 18:3 Δ9Z,12Z,15Z were mainly converted to their corresponding 10-hydro(pero)xy and 8-hydro(pero)xy derivatives (Tab. 1). Interestingly, 18:1 Δ9Z was predominantly oxidized at the C-8, yielding 8-hydro(pero)xy oleic acid (8-H(P)OME), while 18:3 Δ9Z,12Z,15Z was mainly oxidized at the C-10, yielding 10-hydro(pero)xy linolenic acid (10-H(P)OTrE). Additionally, we observed that during incubations of 18:3 Δ9Z,12Z,15Z with PpoC a polar product was formed that eluted at 3.8 min from RP-HPLC. The corresponding tandem MS spectrum (m/z 199) of this compound is shown in Supplemental Fig. 1F and shows the following signals m/z 181 (M --H 2 O), 155 (M --44, probably loss of CO 2 ), 139 (M --60) and 137(M --62). Due to similarity to previously reported MS/MS spectra of 10-ODA [START_REF] Garscha | Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism[END_REF] this product was tentatively assigned as (2E)-decendioic acid.

Identification of volatile products 10-ODA, formed during the incubation of PpoC and 18:2 Δ9Z,12Z , has been described to be a cleavage product of 10-HPODE [START_REF] Labeque | 10-Hydroperoxy-8,12-octadecadienoic acid. A diagnostic probe of alkoxyl radical generation in metal-hydroperoxide reactions[END_REF]. It is known that this compound is formed from β-scission of the hydroperoxide -a reaction which is catalyzed by metals [START_REF] Labeque | 10-Hydroperoxy-8,12-octadecadienoic acid. A diagnostic probe of alkoxyl radical generation in metal-hydroperoxide reactions[END_REF] and by still unidentified fungal lyases [START_REF] Wurzenberger | The formation of 1-octen-3-ol from the 10hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms (Psalliota bispora)[END_REF]. As additional cleavage products volatile octenol-isomers have also been reported [START_REF] Wurzenberger | The formation of 1-octen-3-ol from the 10hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms (Psalliota bispora)[END_REF][29][START_REF] Wurzenberger | Stereochemistry of the cleavage of the 10hydroperoxide isomer of linoleic acid to 1-octen-3-ol by a hydroperoxide lyase from mushrooms (Psalliota bispora)[END_REF]. To elucidate if similar volatile compounds are also formed during the reaction of PpoC with 18:1 Δ9Z , 18:2 Δ9Z,12Z and 18:3 Δ9Z,12Z,15Z , we analyzed the gas phase above the reaction mixture using solid phase micro extraction and GC/MS analysis. Using the NIST library and spectra reported earlier [START_REF] Stumpe | Biosynthesis of C9-aldehydes in the moss Physcomitrella patens[END_REF][START_REF] Wichard | Unprecedented lipoxygenase/hydroperoxide lyase pathways in the moss Physcomitrella patens[END_REF], we were able to identify most of these compounds. We found that during the reaction of PpoC and 18:1 Δ9Z two volatile compounds were formed that could be identified as 1-octanol and 2-undecenal, respectively (Fig. 5A and Tab. 1). The latter compound has been described as an odorant formed from 8-HPOME [START_REF] Grosch | Reactions of hydroperoxides-products of low molecular weight[END_REF]. Volatile compounds that were formed during the reaction of PpoC and 18:2 Δ9Z,12Z were identified as 1-octen-3-ol, 2-octen-1-ol, A c c e p t e d M a n u s c r i p t 3-octenone and 2-octenal, respectively (Fig. 5B and Tab 1). Furthermore, we also detected the formation of two volatile products during incubations of PpoC with 18:3 Δ9Z,12Z,15Z (Fig. 5C). These products could not be reliably identified by using the NIST library. However, incubation with [ 13 C 18 ]-18:3 Δ9Z,12Z,15Z indicated that these products are octadiene-isomers (data not shown). As these volatile compounds were also detected during incubation of 10-hydroperoxy derivatives with empty vector controls, we suggest that these cleavage reactions were mainly caused by non-enzymatic reactions or may be at least catalyzed independently from PpoC. Similar observations have been reported before [START_REF] Garscha | Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism[END_REF].

Peroxidase activity and pH-Optimum

In order to determine if the high amounts of 10-HODE (Fig. 4E) can be formed by PpoC within an enzymatic peroxidase reduction, we performed a peroxidase assay using TMPD as a co-substrate. When purified PpoC was incubated with 100 µM of TMPD and 100 µM of 18:2 Δ9Z,12Z , we were able to follow the characteristic increase at 611 nm for several minutes. A typical progress curve is shown in Supplemental Fig. 2 and is indicative for the peroxidase activity of PpoC. We analyzed the pH-optimum by measuring the initial rates of oxygen consumption for the reaction of PpoC crude extract with 100 µM 18:2 Δ9Z,12Z . The initial rate of oxygen consumption reaches a maximum between pH 7.4 and 8.0. pH values >8.0 led to an rapid decrease in enzymatic activity.

Kinetic properties

Kinetic parameters were determined by measuring the initial rates of oxygen consumption for the reaction with different substrate concentrations using again the Clark-type O 2 -electrode. The reaction was performed with PpoC crude extract; thus we were able to determine kinetic parameters that are independent of the enzyme concentration such as K M . We used sodium salts of 18:1 Δ9Z , 18:2 Δ9Z,12Z and 18:3 Δ9Z,12Z,15Z as substrates and measured the initial rate of oxygen consumption for the different concentration in triplicates. The reaction was performed at pH 7.9 (pH-optimum) and started by the addition of substrate to the oxygen-saturated stirring solution. The K M values are summarized in Tab. 2 and suggest that 18:2 Δ9Z,12Z may be the preferred substrate.

Investigating epoxy alcohol synthase activity of Ppo-enzymes

We detected the formation of 12,13-epoxy-10-hydroxy octadecenoic acid (approx. 3 % of all products formed) when PpoC was incubated with 18:2 Δ9Z,12Z . It has been suggested that this product is formed from 10-HPODE by A. fumigatus PpoC [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. To clarify the enzymatic formation of epoxy alcohols by PpoC, we incubated 10-HPODE with PpoC harbouring bacterial crude extract. Clearly, formation of 12,13-epoxy-10-hydroxy octadecenoic acid could be detected. Next, we also incubated 8-HPODE with PpoC and detected the formation of the corresponding 12,13-epoxy-8-hydroxy octadecenoic acid. The individual RP-HPLC/MS chromatograms of both experiments are shown in Fig. 6. The corresponding MS 2 spectra of both epoxy alcohol derivatives, which are in agreement with those published before [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF] are shown in supplemental Fig. 3A &B. It should be noted, however, that we also detected small amounts of both epoxy alcohols when using cell lysates of empty vector controls (Fig 6A&B). As the detected amounts in control assays were much lower compared to incubations with PpoC, we conclude that the major part of the detected epoxy alcohols are formed by an enzymatic reaction as previously proposed in [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF], but rather by the peroxidase/dioxygenase domain of Ppo enzymes, than by the isomerase domain. To investigate the origin of both oxygen atoms in the epoxy alcohol derivatives, we incubated 18:2 Δ9Z,12Z with bacterial crude lysate under an atmosphere enriched in 18 O 2 . The MS spectrum of the epoxy alcohol product is shown in Fig. 7 and displays two major signals: m/z 311 corresponding to ([ 16 O 2 ]-12,13-epoxy-10-hydroxy octadecenoic acid) and m/z 315 ([ 18 O 2 ]-12,13-epoxy-10-hydroxy octadecenoic acid). As no abundant signal with m/z 313 could be detected, we conclude that the epoxy and the hydroxyl group contain either two atoms of 16 O or two atoms of 18 O. This indicates that both oxygen atoms of the epoxy alcohol group are derived from the same substrate molecule -the fatty acid hydroperoxide. We next examined a potential epoxy alcohol synthase activity of the related PpoA enzyme (purified to homogeneity). Therefore, we incubated 10-HPODE with different concentrations of PpoA. In A c c e p t e d M a n u s c r i p t plotted against PpoA concentrations. We did observe a linear correlation between the PpoA concentrations deployed and product formation or substrate consumption, respectively. Thus, we suggest PpoA to exhibit epoxy alcohol synthase activity with 10-HPODE as a substrate, paralleling the results obtained with PpoC (see above). As reported previously 8-HPODE is predominantly converted to 5,8-DiHODE by the 8-hydroperoxide isomerase P450 domain of PpoA [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. The tandem MS spectrum of this product is shown in Supplemental Fig. 3C. Additionally, we detected traces of 12,13epoxy-8-hydroxy octadecenoic acid formed by PpoA from 8-HPODE.

Site directed mutagenesis of determinants that control site of dioxygenation

Val-385 is a conserved residue in the peroxidase/dioxygenase domain of PpoC, PpoA (Val-328), 7,8linoleate diol synthase (Val-330) and prostaglandin endoperoxide H synthase (Val-349 in sheep PGHS-1). In the latter, Val-349 is known to be important for substrate positioning and the dioxygenase reaction [34]. To compare the oxygenation mechanisms of A. nidulans PpoA and PpoC, we mutated the corresponding amino acids in both enzymes: Leu-385 of PpoC was substituted by either valine or alanine, while Val-328 of PpoA was exchanged either by alanine or leucine. Crude bacterial extract from wild type or mutated PpoC and purified wild type or mutated PpoA was incubated with 18:2 Δ9Z,12Z . The relative amounts of hydroxyl products formed were determined by RP-HPLC/MS and the results are visualized as column diagrams (Fig. 9). For PpoC (Fig. 9A) we observed that substitution of Leu-385 with the smaller residue valine enhanced the formation of 8-HODE by 2-3-fold compared to the wild type enzyme. Reducing the size to the smaller residue like alanine enhanced the formation of 8-HODE by even 3-4 fold as judged by RP-HPLC/MS analysis. We previously reported that we were unable to detect any 10-H(P)ODE formed during incubations of 18:2 Δ9Z,12Z and PpoA by RP-HPLC. Using an improved RP-HPLC/MS approach we now observed the formation of 10-H(P)ODE and especially found that approx. 30 % of the formed hydroxides are 10-HODE (Fig. 9B). The substitution of Val-328 of PpoA by alanine reduced the formation of 8-HODE and increased the production of 10-HODE, whereas substitution with leucine only led to a modest increase in 8-HODE formation compared to the wild type enzyme (Fig. 9B).

Site directed mutagenesis of the catalytic tyrosine

As shown in Fig. 1A Tyr-427 of A. nidulans PpoC is conserved in the enzymes analyzed. Recent mutagenesis studies indicated that this tyrosine residue is important for catalytic activity [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF][34][START_REF] Garscha | Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases[END_REF][START_REF] Koeduka | Catalytic properties of rice aoxygenase. A comparison with mammalian prostaglandin H synthases[END_REF] presumably by generating a tyrosyl radical [START_REF] Su | A protein radical and ferryl intermediates are generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and hydroperoxide isomerase activities[END_REF][START_REF] Smith | Cyclooxygenases: Structural, cellular, and molecular biology[END_REF]. Substitution of PpoC Tyr-427 by Phe (Y427F) led to an inactive enzyme that showed no detectable dioxygenase activity when incubated with 18:2 Δ9Z,12Z . The expression of the Y427F was confirmed by SDS-PAGE analysis (Supplemental Fig. 4).

Site directed mutagenesis of the predicted P450 heme thiolate domain

As shown in Fig. 2B we found that the predicted P450 domain of PpoC does not contain the conserved cysteine residue that is known to be the 5 th heme iron ligand in P450 enzymes [START_REF] Denisov | Structure and Chemistry of Cytochrome P450[END_REF]. Substitution of this cysteine (Cys-1006) in PpoA by alanine led to an enzyme that showed dioxygenase, but no detectable isomerase activity presumably through distortion of the P450 heme moiety [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. As PpoC contains a glycine residue instead of a cysteine at this particular position, we hypothesized that substitution of Gly-1039 by cysteine may reconstitute the heme binding moiety and thereby lead to an enzyme with isomerase activity as it is known for PpoA. However, we did not detect formation of any DiHODE derivative when PpoC_G1039C was incubated with 18:2 Δ9Z,12Z . Moreover, the enzyme showed the same catalytic activity as the wild type enzyme. Moreover, we also incubated the enzyme with 8-HPODE Upon the incubation of PpoC_G1039C with 8-HPODE no DiHODE was detectable.

Radical spin trapping adducts

In order to investigate the formation of carbon centred fatty acid radicals during the reaction of PpoC and 18:2 Δ9Z,12Z we used the method of Koshiishi et al. to scavenge the hypothetical radical with the radical scavenger CmΔP. Recently, we adapted this approach for the PpoA/linoleate system and found the formation of two radical spin trapping adducts which we tentatively assigned as the trapped C-8 and C-10 radical adducts [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF] -a finding that now goes along with our observation that PpoA is also able to form 10-H(P)ODE (Fig. 9B). To test the existence of radical intermediates in the PpoC/linoleate system we performed the trapping reaction with CmΔP using PpoC and 18:2 Δ9Z,12Z . The linoleate allyl radical CmΔP adducts of m/z 463 were identified using RP-HPLC/MS. Two distinct peaks could be detected; a major peak eluting at 28.8 min and a smaller one at 30.8 min [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. We suggest from this result, that the major peak corresponds to the trapped C-8 radical adduct while the smaller peak may correspond to the C-10 radical adduct, in analogy to the PpoA/linoleate system. The MS spectra of both trapping adducts of the PpoC/linoleate system showed the characteristic fragment with m/z 185; this fragment arose from the reduced form of CmΔP through heterolysis of the C-O-N bond [START_REF] Koshiishi | Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition[END_REF]. We also detected trapping adducts with m/z 479 that may correspond to nitroxyl radicallipid epoxy allyl adducts [START_REF] Koshiishi | Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition[END_REF].

DISCUSSION

The present study aimed to characterize A. nidulans PpoC, a putative fatty acid dioxygenase, and to compare it with A. nidulans PpoA -another member of the A. nidulans Ppo-family. The Ppo enzyme family in A. nidulans consists of three dioxygenase genes, PpoA, PpoB and PpoC. Genetic evidence indicated Ppo products to be involved in the regulation of the developmental life cycle of A. nidulans [START_REF] Tsitsigiannis | Endogenous lipogenic regulators of spore balance in Aspergillus nidulans[END_REF]. Interestingly, NCBI conserved domain search predicted that all three protein sequences seem to be fusion proteins because of the presence of two conserved domains: a heme peroxidase/dioxygenase domain in the N-terminal region of the enzymes and a cytochrome P450 region in the C-terminal part. Recently, we showed that the bifunctional activity of PpoA is indeed related to the presence of both domains: within the peroxidase/dioxygenase domain of PpoA the (8R)-dioxygenase reaction takes place while in the P450 domain the (8R)-hydroperoxide isomerisation is accomplished [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. Although PpoC is predicted to be a fusion protein as well having a similar C-terminal P450 domain, we found on the basis of sequence alignments, that the crucial cysteine residue is not conserved in its sequence (Fig. 2B). In P450 enzymes the corresponding residue acts as the 5 th heme iron ligand and mutation of this cysteine leads to an inactive enzyme [START_REF] Yoshioka | Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam[END_REF]. Therefore, it appeared doubtful that this domain is functional in PpoC and has catalytic activity. To analyze the influence of this changed domain structure we isolated PpoC cDNA, expressed the enzyme in E. coli and investigated its biochemical properties.

Our results indicate that both fusion proteins, PpoA and PpoC, have many properties in common, but there are also essential differences that are most likely caused by the differences in their C-terminal P450 domain. PpoA and PpoC resemble 7,8-linoleate diol synthase tetrameric heme proteins that catalyze the oxidation of 18:2 Δ9Z,12Z . While PpoA predominantly oxidizes 18:2 Δ9Z,12Z at the C-8, yielding 8-HPODE, PpoC mainly performs oxygenation at the C-10 forming 10-HPODE. These dioxygenase reactions are both catalysed within the peroxidase/dioxygenase domain of the fusion proteins (Fig. 9). Sequence alignments showed that PpoA and PpoC contain the conserved tyrosine within their heme peroxidase/dioxygenase domain (Tyr-374 in PpoA and Tyr-427 in PpoC) that is also found in 7,8-linoleate diol synthase (Tyr-376), sheep prostaglandin endoperoxide H synthase-1 (Tyr-385) and sheep prostaglandin endoperoxide H synthase-2 (Tyr-370). Previous studies showed that dioxygenase activity was reduced or even abolished in these enzymes when this tyrosine residue was mutated [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]34,[START_REF] Garscha | Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases[END_REF]. As also reported previously for PpoA, we now provide evidence that substitution of this tyrosine by phenylalanine also abolished dioxygenase activity of PpoC. Thus, we conclude that PpoC also uses a tyrosyl radical for the abstraction of the hydrogen from the C-8 of 18:2 Δ9Z,12Z and forms a carbon centred radical at this position. The unpaired electron is delocalized and can migrate from the C-8 to the C-10 by shifting the C-9-C-10 double bond to C-8-C-9. The formation of carbon centred substrate radicals at the C-8 and C-10 was also supported by experiments in which we trapped these radicals using the radical scavenger CmΔP and as reported before for other enzymes [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF][START_REF] Koshiishi | Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition[END_REF]. The data so far led us to ask what the amino acid determinants are that direct the oxygenation at C-8 and C-10, respectively. Recently, Garscha et al. reported the recombinant expression of a PpoC from A. fumigatus that catalyzes a similar reaction as PpoC [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. By substituting Leu-384 and Val-388 with larger and smaller amino acids they could show that these residues act as determinants that direct oxygenation either at C-8 or C-10 of 18:2 Δ9Z,12Z [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. As similar substitutions of the homologue residue in G. graminis 7,8-linoleate diol synthase (Val-330) showed a related effect, they developed a general model for the oxygenation by fungal 8-dioxygenases and 10-dioxygenases, respectively. This model proposed that Leu-384 shields the C-8 of 18:2 Δ9Z,12Z in the active site of (10R)-dioxygenase or PpoC [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. To gain further insights into the oxygenation mechanisms, we also mutated the related Val-328 (in PpoA) and Leu-385 (in PpoC) residues in the Ppo-enzymes from A. nidulans. This experiment enabled for the first time the direct comparison of two dioxygenases present in the same species and A c c e p t e d M a n u s c r i p t with high amino acid sequence similarity but different product specificities. For the substitution of PpoC Leu-385 by alanine and valine we found similar effects as reported [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF] that may also indicate that Leu-385 shields the C-8 of 18:2 Δ9Z,12Z . However, the substitution of Val-328 in PpoA by leucine and alanine conversely indicated that this residue might shield C-10 rather than C-8. This effect may be explained by a different positioning or even by a different orientation of the fatty acid in the active site of both enzymes, leading to the conclusion that our results in contrast to those of Garscha et al. [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF] indicate that there either is no general model for oxygenation by these fungal dioxygenases or the role of the mutated amino acids is as yet unclear. Clearly, a crystal structure is needed to further address this question. A further explanation may lay in the fact that the isomerase activity of recombinant 7,8-linoleate diol synthase is very weak and, therefore, 8-H(P)ODE can accumulate. This accumulation might influence the equilibrium in a different way compared to PpoA where almost all 8-HPODE is immediately converted to 5,8-DiHODE. We further investigated the downstream products of the dioxygenase reaction of PpoC. 10-HPODE that was formed during incubations of PpoC and 18:2 Δ9Z,12Z was mainly converted into 10-HODE, which was formed by an enzymatic peroxidase reaction of PpoC within the C-terminal heme peroxidase/dioxygenase domain. Another product of this domain was 10-KODE. However, 10-HPODE appeared to be unstable on its own and 10-ODA and volatile compounds like 2-octen-1-ol, 2octenal, 3-octanone and 1-octen-3-ol were formed most likely by chemical fragmentation from this PpoC product. The latter compound has also been reported to be formed from 18:2 Δ9Z,12Z by homogenates of Lentinus decadetes [START_REF] Matsui | Linoleic acid 10-hydroperoxide as an intermediate during formation of 1-octen-3-ol from linoleic acid in Lentinus decadetes[END_REF], Psalliota bispora [START_REF] Wurzenberger | The formation of 1-octen-3-ol from the 10hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms (Psalliota bispora)[END_REF][29][START_REF] Wurzenberger | Stereochemistry of the cleavage of the 10hydroperoxide isomer of linoleic acid to 1-octen-3-ol by a hydroperoxide lyase from mushrooms (Psalliota bispora)[END_REF] and Tricholoma matsutake [START_REF] Akakabe | Stereochemical correlation between 10hydroperoxyoctadecadienoic acid and 1-octen-3-ol in Lentinula edodes and Tricholoma matsutake mushrooms[END_REF]. Although the reaction has been discovered 25 years ago, the enzyme involved in formation of these compounds known as mushroom flavour was never identified from neither of these sources. Interestingly, at first an independent metabolic reaction leading to formation of the same volatiles 2octen-1-ol and 1-octen-3-ol was described for the moss Physcomitrella patens [START_REF] Wichard | Unprecedented lipoxygenase/hydroperoxide lyase pathways in the moss Physcomitrella patens[END_REF]. Here a bifunctional lipoxygenase uses the C-20 fatty acid arachidonic acid and the C-8 alcohols are formed via the (12S)-hydroperoxide of this fatty acid [START_REF] Senger | A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens[END_REF]. As a further product 12,13-epoxy-10-hydroxy octadecenoic acid was formed during the reaction of PpoC and 18:2 Δ9Z,12Z . This product has also been reported to be formed by PpoC from A. fumigatus [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. It was suggested that this enzyme has epoxy alcohol synthase activity and converts specifically 10-HPODE to 12,13-epoxy-10-hydroxy octadecenoic acid in analogy to the isomerisation reaction that is performed with 8-HPODE by 7,8-linoleate diol synthase [START_REF] Garscha | Leucine/Valine Residues Direct Oxygenation of Linoleic Acid by (10R)-and (8R)-Dioxygenases: EXPRESSION AND SITE-DIRECTED MUTAGENESIS OF (10R)-DIOXYGENASE WITH EPOXYALCOHOL SYNTHASE ACTIVITY[END_REF]. Indeed, by incubating purified 10-HPODE with PpoC bacterial crude extract we confirmed the enzymatic conversion of this substrate to 12,13-epoxy-10-hydroxy octadecenoic acid. Thus, we showed for the first time that 10-HPODE is in fact the precursor of 12,13-epoxy-10-hydroxy octadecenoic acid. Additionally, we found that PpoC can also convert 8-HPODE into 12,13-epoxy-8-hydroxy octadecenoic acid but not into 5,8-DiHODE. This finding underlines that a functional P450 domain, as in case of PpoA, is essential for an isomerisation reaction but not for epoxy alcohol synthase activity. As 7,8-linoleate diol synthase displays its bifunctionality by conversion of 18:2 Δ9Z,12Z to 8-HPODE followed by the isomerisation yielding 7,8-DiHODE, we next investigated if purified PpoA also harboured a similar epoxy alcohol synthase side activity besides the described dioxygenase and isomerase activity. Incubations of purified PpoA and 8-HPODE led mainly to the formation of 5,8-DiHODE by the 8-hydroperoxide isomerase P450 domain as reported. As we also detected low amounts (< 1 % of total reaction products) of 12,13-epoxy-8-hydroxy octadecenoic acid during these incubations we concluded that like PpoC also PpoA harbours a epoxy alcohol synthase side activity. This was further confirmed by incubations of PpoA with 10-HPODE (Fig. 8). In contrast to 8-HPODE this substrate was not converted into a dihydroxy fatty acid but rather transformed into 12,13-epoxy-10-hydroxy octadecenoic acid. The finding that the amount of 12,13-epoxy-10-hydroxy octadecenoic acid showed a linear dependency from concentrations of the PpoA used in the experiment additionally indicate an enzymatic reaction. However, as we also found production of epoxy alcohols in negative controls in minor amounts, we suggest that epoxy alcohol derivatives can also be formed from 10-HPODE and 8-HPODE under the used assay conditions. As the isomerase activity of the bifunctional PpoA is located in the P450 domain and PpoC seems to contain an inactive P450 domain, due to the lack of a highly conserved cysteine residue, we tried to obtain a bifunctional PpoC by mutation analysis. To reconstitute the P450 heme binding moiety and to obtain functional 8-hydroperoxide isomerase domain we substituted of Gly-1039 with cysteine and incubated the enzyme with 18:2 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2009 The Authors Journal compilation © 2009 Portland Press Limited activity was retrieved. This might indicate that several more residues in the PpoC sequence are important for correct binding of heme and a correct folding of the P450 domain. However, for conclusive identification of further essential amino acids constituting a functional P450 motif a crystal structure is required. A domain switching approach may also be suitable to address this question in the future, but the lack of information about the crystal structure of the enzyme is hindering further investigations in that direction as well. Taken together, our results show that PpoA and PpoC are fusion proteins that employ similar mechanisms to catalyze the formation of 8-and 10-hydroperoxy fatty acids. In this context the leucine/valine residues that have been reported recently for A. fumigatus PpoC and G. graminis 7,8linoleate diol synthase seem also to determine the position of oxygenation of 18:2 Δ9Z,12Z , i.e. C-8 and C-10, of dioxygenases in A. nidulans, but a general mechanism how these amino acid residues influence the site of oxygenation remains unclear. PpoC and PpoA both have an epoxy alcohol synthase side activity and can convert 8-HPODE and 10-HPODE into the corresponding epoxy alcohols. However, this is employed by the peroxidase/dioxygenase domain of these enzymes. This is further supported by the fact that PpoA converts 8-HPODE mainly into 5,8-DiHODE, while PpoC cannot perform a similar reaction, probably due to a degenerated P450 heme thiolate domain.

FIGURE-LEGENDS

FIGURES Figure 1:

Psi factor formation in A. nidulans and A. fumigatus. Ppo enzymes abstract a hydrogen atom from the C-8 of the backbone of unsaturated C-18 fatty acids and form a carbon centred radical at this position -a reaction that is presumably accomplished via a tyrosyl radical mechanism within the N-terminal fatty acid heme peroxidase/dioxygenase domain. In case of enzymes belonging to PpoA subfamily molecular oxygen attacks the C-8 centred radical and forms (8R)-HPODE. This intermediate product is either used as a substrate for peroxidase reaction, yielding 8-HODE, or it serves as a substrate for the 8-hydroperoxide isomerase reaction, yielding mainly 5,8-DiHODE. The isomerase reaction is believed to be accomplished by the C-terminal P450 heme thiolate domain of PpoA [START_REF] Brodhun | Identification of PpoA from Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a cytochrome P450[END_REF]. Thirdly the fatty acid hydroperoxide is reduced to 8-HODE or transformed to small amounts of 12,13-epoxy-8-hydroxy octadecenoic acid, respectively. In case of PpoC enzymes the carbon centred radical migrates to C-10 and molecular oxygen is inserted at this position, yielding 10-HPODE. This product appears to be unstable and either oxidizes to 10-KODE or decomposes to 10-ODA and volatile compounds as 1-octen-3-ol. Furthermore, 10-HPODE can be reduced to 10-HODE or transformed to small amounts of 12,13-epoxy-10-hydroxy octadecenoic acid, respectively.

Figure 2:

Partial alignment of the predicted N-terminal heme peroxidase/dioxygenase domain and C-terminal P450 domain from PpoC with sequences from different fatty acid heme dioxygenases and P450 enzymes. A. Partial amino acid alignment of the predicted heme peroxidase/dioxygenase domain from A. nidulans PpoC (XP_662632; AnPpoC) with A. fumigatus PpoC (ABV21633; AfPpoC), A. nidulans PpoA (XP_659571; AnPpoA), G. graminis 7,8-linoleate diol synthase (Q9UUS2.1; GgLDS), sheep prostaglandin endoperoxide H synthase-1 (P05979.2) and sheep prostaglandin endoperoxide H synthase-2 (P79208.1). The catalytic tyrosine as well as the proximal and distal histidines, which are conserved in the six sequences and have been identified before [START_REF] Garscha | Critical amino acids for the 8(R)-dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases[END_REF][START_REF] Picot | The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1[END_REF] are shaded black. Additionally, leucine/valine residues that are responsible for direction of oxygenation in A. fumigatus PpoC and G. graminis 7,8-linoleate diol synthase are shaded black. B. Partial amino acid alignment of the predicted P450 heme thiolate domain from A. nidulans PpoC with known P450 enzymes [CYP450_119 (1F4T:B), CYP450_cin (1T2B:B), CYP450_130 (2UUQ:A), CYP450_nor (1JFB:A), CYP4503A4 (1TQN:A), CYP4502C9 (1R9O:A)] and predicted CYP450 domains from different Ppo-enzymes [A. fumigatus PpoC (ABV21633), A. nidulans PpoA (XP_659571) and A. fumigatus PpoA (XP_751750)]. Amino acids matching the P450 consensus sequence according to the PROSITE database are shaded grey. Additionally the cysteine responsible for the coordination of heme iron is shaded black. The sequences were aligned using the ClustalW algorithm. . The products were identified with SPME, GC/MS and by using the NIST library and spectra reported earlier [START_REF] Stumpe | Biosynthesis of C9-aldehydes in the moss Physcomitrella patens[END_REF][START_REF] Wichard | Unprecedented lipoxygenase/hydroperoxide lyase pathways in the moss Physcomitrella patens[END_REF]. Incubations of PpoC with 18:1 Δ9Z led to the production of 1-octanol and 2-undecenal, while during incubations with 18:2 Δ9Z,12Z predominantly 1octen-3-ol, 2-octen-1-ol, 2-octenal and 3-octenon were formed. 18:3 Δ9Z,12Z,15Z was converted by PpoC into two volatile products which could not be identified using the NIST library. By experiments with [ 13 C 18 ]-labelled 18:3 Δ9Z,12Z,15Z these products were tentatively assigned as octadiene isomers. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

GTACGCAAATATTATCCTTAAUGUGATTATGCACGAACGATTTTGAATATAAA

Biochemical

  Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096

Biochemical

  Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096

  Fig. 8 the relative amounts of 12,13-epoxy-10-hydroxy octadecenoic acid, 10-ODA and 10-HPODE are Biochemical Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096

Biochemical

  Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096

Figure 3 :

 3 Figure 3: SDS-PAGE at different stages of purification (A) and UV/vis absorption of purified PpoC (B). A. Aliquots after each purification step were analysed by 8 % SDS-PAGE. Lane 1, crude cell extract; lane 2, after Source 30Q; lane 3, after Sephadex S200 (60/20), lane 4, after MonoQ (10/10). B. A spectrum of the native enzyme in 50 mM HEPES (pH 7.4) is shown. The UV/vis spectrum of the purified enzyme showed absorption maxima at 403 nm (γ, Soret) indicating the presence of heme. Additionally two weaker maxima at approximately 530 nm and 630 nm are indicated by arrows.

Figure 4 :

 4 Figure 4:RP-HPLC/MS analysis of metabolites formed from 18:2 Δ9Z,12Z by recombinant PpoC. RP-HPLC/MS analysis after 30 min incubation of 18:2 Δ9Z,12Z with PpoC in 50 mM Tris buffer (pH 8.0) and extractive isolation. RP-HPLC/MS analysis of (A) 10-HPODE and 12,13-epoxy-10hydroxy octadecenoic acid (m/z 311; full scan), (B) 8-HODE and 10-HODE (m/z 295; full scan), (C) 10-KODE (m/z 293; full scan) and (D) 10-ODA (m/z 183; full scan). As reported previously[START_REF] Garscha | Identification of dioxygenases required for Aspergillus development: Studies of products, stereochemistry, and the reaction mechanism[END_REF], 10-HPODE decomposed in the mass spectrometer into 10-ODA which co-elutes with 10-HPODE at 20.9 min, while pre-formed 10-ODA elutes at 3.8. E. Relative amounts of products formed during the incubation. Mean values and standard deviations were calculated from three different experiments.

Figure 5 :

 5 Figure 5:GC/MS-analysis of volatile compounds of PpoC and different fatty acid substrates. Volatile compounds formed during the incubation of PpoC with (A) 18:1 Δ9Z , (B) 18:2 Δ9Z,12Z and (C)18:3 Δ9Z,12Z,15Z . The products were identified with SPME, GC/MS and by using the NIST library and spectra reported earlier[START_REF] Stumpe | Biosynthesis of C9-aldehydes in the moss Physcomitrella patens[END_REF][START_REF] Wichard | Unprecedented lipoxygenase/hydroperoxide lyase pathways in the moss Physcomitrella patens[END_REF]. Incubations of PpoC with 18:1 Δ9Z led to the production of 1-octanol and 2-undecenal, while during incubations with 18:2 Δ9Z,12Z predominantly 1octen-3-ol, 2-octen-1-ol, 2-octenal and 3-octenon were formed. 18:3 Δ9Z,12Z,15Z was converted by PpoC into two volatile products which could not be identified using the NIST library. By experiments with [ 13 C 18 ]-labelled 18:3 Δ9Z,12Z,15Z these products were tentatively assigned as octadiene isomers.

Figure 6 :

 6 Figure 6: Conversion of 8-HPODE and 10-HPODE to epoxy alcohols by PpoC. Shown are the results from RP-HPLC/MS analysis after incubation of 10-HPODE (A & C) and 8-HPODE (B & D) with empty vector controls (A & B) and recombinant PpoC bacterial crude extract (C & D) after 40 min incubation at room temperature.

Figure 7 :

 7 Figure 7:Mass spectrometric analysis of 12,13-epoxy-10-hydroxy octadecenoic acid formed from 18:2 Δ9Z,12Z by PpoC under18 O 2 . RP-HPLC/MS analysis of the 12,13-epoxy-10-hydroxy octadecenoic acid obtained after incubation of 18:2 Δ9Z,12Z with recombinant PpoC bacterial crude extract under enriched 18 O 2 atmosphere.

Figure 8 :

 8 Figure 8: Conversion of 10-HPODE to 12,13-epoxy-10-hydroxy octadecenoic acid and 10-ODA as a function of PpoA concentration. The reaction system contained 500 µM 10-HPODE and various concentration of purified PpoA in 50 µM HEPES (pH 7.4). The reaction was terminated after 40 min.

Figure 9 :

 9 Figure 9: Relative amounts of 8-HODE formed by PpoA_WT, PpoC_WT and their corresponding mutants. A. Relative amounts of 8-HODE formed by recombinant PpoC_WT, PpoC_L385A and PpoC_L385V bacterial crude extract in 50mM Tris/HCl (pH 8.0). B. Relative amounts of 8-HODE formed by purified PpoA_WT, PpoA_V328A and PpoC_V328L in 50 mM HEPES (pH 7.4). Mean values and standard deviations were calculated from three different experiments.

  Figure 2 A Alignment Peroxidase/dioxygenase domain AnPpoC YQYRSADGSNNNPTLPWLGAANTAYARSIEPLTVQPGGLPDAGLVFDTLFARQ--KFTPH 237 AfPpoC YAYRSADGSNNNPTLPRLGAANTLYARTIPPLIIQPGGLPDPGLVFDTLFARQ--TFKPH AnPpoA SMHRKADGSGNNRFWPQLGAAGSAYARSVRPKTMQSPSLPDPETIFDCLLRRK--EYREH GgLDS FSFREPDGSNNNIHLPSLGAANTPYARSTKPLVFQNPNPPDPATIFDTLMVRDPAKFRPH SheepPGHS1 YISWESFS---NVSYYTRILPSVPKDCPTPMGTKGKKQLPDVQLLAQQLLLRR--EFIPA SheepPGHS2 YKSWEAFS---NLSYYTRALPPVADDCPTPMGVKGNKELPDSKEVLEKVLLRR--EFIPD .. . * . . ** : : :: * : AnPpoC PNKVSSLFFDWASLIIHDIFQTDYRDYN--KNKTSAYLDLAILYGDVQEEQDLVRTHKDG 295 AfPpoC PNKVSSVFFYWASLIIHDIFQTDYKNPN--MNKTSGYLDLSILYGDVQEEQNLIRTFKDG AnPpoA PNKISSVLFYLASIIIHDLFQTDPKDNS--VSKTSSYLDLSPLYGNNQDEQNLVRTFKDG GgLDS PNKISSMLFYLATIITHDIFQTSPRDFN--INLTSSYLDLSPLYGRNHDEQMAVRTGKDG SheepPGHS1 PQGTNILFAFFAQHFTHQFFKTSGKMGPGFTKALGHGVDLGHIYGDNLERQYHLRLFKDG SheepPGHS2 PQGSNMMFAFFAQHFTHQFFKTDHKRGPGFTRGLGHGVDLNHIYGETLDRQHKLRLFKDG *: . :: * : *::*:*. : . :** :** :.* :* *** AnPpoC KLKPDSFS----------EPRLQAFPAACCVLLVMLNRFHNYVVEELAAINENGRFTKPS 345 AfPpoC KLKPDSFS----------EPRLQAFPATCCVLMVMLNRFHNYAVEQLAAINENGRFTKPA AnPpoA KLKPDCFA----------TKRVLGFPPGVGVLLIMFNRFHNYVVDQLAAINECGRFTKP-GgLDS LLKPDTFS----------SKRVIGFPPGVGAFLIMFNRFHNYVVTQLAKINEGGRFKRP-SheepPGHS1 KLKYQVLDGEVYPPSVEQASVLMRYPPGVPPERQMAVGQEVFGLLPGLMLFSTIWLREHN SheepPGHS2 KLKYQVIGGEVYPPTVKDTQVEMIYPPHIPENLQFAVGQEVFGLVPGLMMYATIWLREHN ** : : :*. : . : : : : . AnPpoC PDLPEEQAKKAWAKYDEDLFQTGRLITCGLFINITLYDYLRTIVNLNRVNSTWCLDPRAQ 405 AfPpoC DNLSEEEAKKAWAKYDEDLFQTGRLITCGLYINITLYDYLRTIVNLNRTNSTWCLDPRAQ AnPpoA ----DESNVDEYAKYDNNLFQTGRLVTCGLYANIILKDYVRTILNINRTDSTWSLDPRME GgLDS ---TTPDDTAGWETYDNSLFQTGRLITCGLYINIVLGDYVRTILNLNRANTTWNLDPRTK SheepPGHS1 -RVCDLLKEEHPTWDDEQLFQTTRLILIGETIKIVIEEYVQHLS---GYFLQLKFDPELL SheepPGHS2 -RVCDILKQEHPEWGDEQLFQTSRLILIGETIKIVIEDYVQHLS---GYHFKLKFDPELL *:.**** **: * :* : :*:: : :**. AnPpoC MEG---SAT-PAGLGNQCSVEFNLAYRWHSAISANDEKWTEKVYEELI-GKPGSEISTQE 460 AfPpoC MEG---SHTAPSGLGNQCSVEFNLAYRWHSATSATDEKWTEDVYERLM-GKPASEVSMTE AnPpoA MKDGLLGEAAAMATGNQVSAEFNVVYRWHACISKRDEKWTEDFHREIMPGVDPSTLSMQD GgLDS EGKSLLSKPTPEAVGNQVSVEFNLIYRWHCTISERDDKWTTNAMREALGGQDPATAKMED SheepPGHS1 FRA-------QFQYRNRIAMEFNHLYHWHPLMPNSFQ-------------VGSQEYSYEQ SheepPGHS2 FNQ-------QFQYQNRIASEFNTLYHWHPLLPDTFN-------------IEDQEYSFKQ *: : *** *:** . : . : AnPpoC LLMGLGKYGASLPKDPSQRTFAGLKRQEDGTFKDEELVNILTSAIEDVAGSFGARNVPKV 520 AfPpoC LLMGLGKYQAELPKDPSKRTFADLERQADGRFKDEDLVNLLVNAVEDVAGSFGARNVPKV AnPpoA FVAGLGRWQAGLPQEPLERPFSGLQRKPDGAFNDDDLVNLFEKSVEDCAGAFGASHVPAI GgLDS VMRALGMFEKNIPDEPEKRTLAGLTRQSDGAFDDTELVKILQESIEDVAGAFGPNHVPAC SheepPGHS1FLFNT----SMLVDYGVEALVDAFSRQRAGRIG-------------------GGRNFDYH SheepPGHS2 FLYNN----SILLEHGLTQFVESFTRQIAGRVA-------------------GGRNVPIA .: : . . : *: * . * :.

  Figure 3

  Figure 4

  Figure 5

  Figure 6

  Figure 8

  Figure 9

  Biochemical Journal Immediate Publication. Published on 30 Oct 2009 as manuscript BJ20091096 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091096

Δ9Z,12Z and 8-HPODE. No functional 8-hydroperoxide isomerase

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

The authors wish to thank Sabine Freitag and Theres Riemekasten for expert technical assistance and Nambirajan Govindarajan for initial cloning experiments. Critical reading of the manuscript by Dr. I. Heilmann is also gratefully acknowledged. Assignment of PpoC and comments on the PpoC P450 sequence by Dr. D.R. Nelson are also gratefully acknowledged.

Fermentas (St. Leon-Roth, Germany).

FUNDING

This work was supported by the German Research Foundation in the framework of the IRTG 1422.

ABBREVIATIONS FOOTNOTES

The abbreviations used are: CmΔP, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl; DiHODE, dihydroxy octadecadienoic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid; H(P)OME, hydro(pero)xy octadecenoic acid; H(P)ODE, hydro(pero)xy octadecadienoic acid; H(P)OTrE, hydro(pero)xy linolenic acid; 10-KODE, (8E,12Z)-10-oxo-8,12-octadecadienoic acid; ODA, 10-oxo-8-decenoic acid; Ppo, psi producing oxygenase; ppo, gene coding for Ppo; Psi,

TABLES TABLE 1: Oxygenation products of A. nidulans PpoC.

Products were identified using RP-HPLC/MS 2 analysis and by detection of characteristic signals (8hydroperoxide derivative with m/z 157 (cleavage between C-8 and C-9) and 10-hydroperoxide derivative with m/z 183 (cleavage between C-10 and C-11, as also shown in Supplemental Figure 2). Volatile products were extracted by solid phase micro extraction and identified by GC/MS analysis using the NIST database (Figure 6).

Substrate Hydroperoxide products

Hydroperoxide derived products

Volatile products Kinetic analysis was performed with a Clark-type O 2 -electrode. Initial time dependent rates of oxygen consumption were measured with different substrate concentrations and K M -values were determined by fitting seven data points to the Michaelis-Menten-equation using the software Origin 6.0. To prevent mycel formation the sodium salts of each fatty acid was used as a substrate for the kinetic analysis. Mean values were calculated from three different experiments.

Substrate K M -value [µM]

18:1 Δ9Z 13.1 ± 1.7 18:2 Δ9Z,12Z

3.6 ± 0.97 18:3 A c c e p t e d M a n u s c r i p t

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