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 (18)O2 and (16)O2 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 Asn(965) with Val in the conserved AsnGlnXaaGln sequence revealed that Asn(965) 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 function of the second gene, MGG_10859, 5 is unknown, but MGG_10859 is upregulated during appressorium formation and downregulated by gene deletion of PMK1 6 ( 30 ). Circumstantial evidence suggests that MGG_10859 may also be regulated by PKA. A mutant strain of M. oryzae (DA-99) with constitutive PKA activity ( ⌬ mac1 sum1-99 ) oxidized linoleic acid to a new profi le of metabolites, likely due to expression of linoleate 10 R -DOX with MGG_10859 as a possible but unproven candidate gene ( 29 ). The PMK1 -controlled expression of MGG_10859 and the distribution of MGG_10859 homologs in devastating plant pathogens [ Fig. 1B ; Gaeumannomyces graminis (Takeall of wheat), C. graminicola (Anthracnose of maize), and Fusarium oxysporum (Panama disease of banana)] indicate a pathophysiological function, which is noteworthy.
The fi rst objective of the present report was to express the two putative DOX-CYP fusion proteins of M. oryzae , MGG_10859 and MGG_13239, because the former could be the fi rst member of a new subfamily ( Fig. 1B ). The second objective was to reexamine the products formed by the mutant strain DA-99 with constitutive PKA activity ( ⌬ mac1 sum1-99 ) of M. oryzae with regard to the catalytic activities of MGG_10859 and MGG_13239. Finally, we also expressed the MGG_10859 homolog of F. oxysporum , FOXB_03425, 7 to determine whether MGG_10859 and this protein possess the same enzyme activities.
Genome sequences of pathogenic fungi are being published at an increasing rate ( 18,19 ). The catalytic activities of emerging DOX-CYP fusion proteins have not yet been fully determined. Putative new DOX-CYP subfamilies can be described by amino acid identity and by the linked and specifi c dual catalytic activities. In contrast, mammalian P450 families and subfamilies are defi ned by >40% and 55% amino acid identity, respectively, and not by their catalytic activities due to the functional redundancy within subfamilies. DOX-CYP fusion enzymes of different subfamilies can generally be aligned with 35% to 50% sequence identity, whereas enzymes of subfamilies with identical catalytic activities usually align with 60% or higher sequence identity. Based on the latter, a tentative new DOX-CYP subfamily could be identifi ed in several genera of the top 10 plant pathogens (e.g., Magnaporthe , Colletotrichum , and Fusarium ) ( Fig. 1B ) ( 20 ).
Wheat, rice, maize, and banana are staple crops, and rice feeds half of the world's population. Fungi constitute a constant threat to this food supply. Magnaporthe oryza 3 causes rice blast disease, which annually reduces the rice harvest by 10% to 30% ( 21 ). Colletotrichum graminicola infects maize at annual costs in Northern America on the order of 1 billion US$. Both fungi share a common infection process. Their conidia develop into an injection apparatus, the appressorium, required for blast cuticle penetration ( 21,22 ). M. oryzae is the prototype organism for studying this process. Its appressorium accumulates lipid bodies, trehalose, and glycerol from the conidium, and the latter generates the turgor pressure for blast penetration. The catalytic subunit of cAMP-dependent protein kinase A (PKA), mitogen-activated protein kinase PMK-1 (PMK1), and triacylglycerol lipases take part in mobilization of fatty acids of lipid bodies in support of the infection process (23)(24)(25)(26)(27).
The genome of M. oryzae is sequenced ( 28 ). It is estimated to contain about 11,000 genes, and 2 of them code for DOX-CYP fusion proteins. One of them, MGG_13239, 4 was previously identifi ed as 7,8-LDS by gene deletion ( 29 ). The cells were harvested by centrifugation (13,000 rpm, 4°C, 25 min) and sonicated (Bioruptor Next Gen, 10 × 30 s, 4°C). Cell debris was removed by centrifugation, and the supernatants were used immediately or frozen at Ϫ 80°C until needed. Each protein was expressed in at least three independent expression experiments.

Site-directed mutagenesis of recombinant proteins
Site-directed mutagenesis was performed according to the Quick Change protocol (Stratagene) with 10 ng of the pUC57 constructs as templates and Pfu DNA polymerase (16 cycles). PCR products were incubated with Dpn I (37°C, 2 h) to digest maternal DNA. Gel electrophoresis confi rmed amplifi cation of one distinct PCR product, which was then used for transformation of E. coli (NEB5 ␣ ) cells by heat-shocking. All mutations were confi rmed by vernolic acids was performed with methylene blue ( 32 ). 18 O 2 gas (97%) was obtained from Isotec (Sigma-Aldrich). SepPak columns (silicic acid or C 18 ) were from Waters.

Expression of recombinant proteins
The open reading frames of MGG_13239, MGG_10859, and FOXB_03425 in pUC57 vectors were transferred to pET101D-TOPO vectors by PCR technology according to Invitrogen's instructions (all primers are listed in supplementary Table I). Competent E. coli (BL21) cells were transformed with the expression constructs by heat-shocking. Cells were grown until A 600 of 0.6-0.8 in 2xYT medium prior to addition of 0.1 mM isopropyl ␤ -D -1-thiogalactopyranoside to induce protein expression. After 5 h under moderate shaking ( ‫ف‬ 100 rpm) at room temperature, the collision energy at 35 (arbitrary scale), and the tube lens varied between 90 and 120 V. Prostaglandin F 1 ␣ was infused for tuning. Samples were injected manually (Rheodyne 7510) or by an autosampler (Surveyor Autosampler Plus, ThermoFisher).
Hydrogenation was performed with Pd/C in methanol and a small stream of hydrogen for 90 s. The catalyst was removed by fi ltration through Na 2 SO 4 . Chlorohydrin adducts of epoxides were prepared by reaction with 40 l of methoxamine HCl in pyridine (20 mg/ml; 60°C, 1 h) ( 34 ). Water was added, and the products were purifi ed on a C 18 cartridge (SepPack/C 18 ) column before analysis by HPLC/MS 2 .

Bioinformatics
The ClustalW algorithm was used for sequence alignments (Lasergene, DNASTAR Inc.), and the MEGA6 software for construction of phylogenetic trees with bootstrap tests of the resulting nodes ( 35 ). The distance between the branches is indicative of the expected number of substitutions per amino acid position.
Steric analysis showed that 10 R -HPODE was formed with high stereoselectivity ( Fig. 2C ). This metabolite was apparently transformed to an epoxy alcohol, which was identifi ed as 12 S (13 R )-epoxy-10 R -hydroxy-8( E )-octadecenoic acid as described subsequently. The epoxy alcohol was formed from 10 R -HPODE by an intramolecular rearrangement mechanism as judged by experiments performed under a mixture of 16 O 2 and 18 O 2 gas. The anions of 12(13)-epoxy-10-hydroxy-8( E )-octadecenoic acid, which were obtained under this condition, had m / z values of either 311 or 315, but not 313 ( Fig. 2D ). This demonstrates that the oxygen atom in the epoxy group of one molecule must have evolved from the hydroperoxy group of the same molecule. MGG_10859 will therefore be referred to as 10 R -DOX-EAS. sequencing before subcloning to pET101D-TOPO vectors described previously. All primers are listed in supplementary Table II.

Growth of M. oryzae (Guy11) and strain DA-99
M. oryzae Guy11 was grown in liquid culture for 5-16 days in complete medium at 22°C with a cycle of 16 h of fl uorescent light (True-light) followed by 8 h of darkness ( 29 ). The genetically modifi ed strain DA-99 of M. oryzae [ ⌬ mac1 sum1-99 ( 25 )] was grown in the same way. Mycelia (0.5-20 g wet weight) were ground to a powder in liquid nitrogen and stored at Ϫ 80°C. Aliquots were thawed and homogenized, and the supernatants were assayed for enzyme activity with 18:2 n -6 as substrate as described previously. Nitrogen powders of both strains were assayed in parallel at different time points of growth. Each experiment was performed at least in duplicate. Conidia were harvested from mycelia, which had grown for 8-12 days on complete medium agar plates with a glass spreader and by rinsing with distilled water.

LC/MS analysis
Reversed phase (RP)-HPLC with MS 2 analysis was performed with a Surveyor MS pump (ThermoFisher) and an octadecyl silica column (5 µm; 2.0 × 150 mm; Phenomenex), which was eluted at 0.3 ml/min with methanol-water-acetic acid, 800:200:0.05, or 750:250:0.05. The effl uent was subject to ESI in a linear ion trap mass spectrometer (LTQ, ThermoFisher). The heated transfer capillary was set at 315°C, the ion isolation width at 1.5 amu, the We next examined the fragmentation of the epoxy alcohol with regard to the signals at m/z 211, 193, and 181 by recording the MS 2 spectra of three derivatives: i ) the hydrogenated epoxy alcohol, ii ) the uniformly 13 C-labeled epoxy alcohol, and iii ) and the epoxy alcohol formed under 18  These results suggest that the signal at m/z 211 (A Ϫ -(18+82)) could be due to loss of water and C 6 H 10 from the carboxylate anion (A Ϫ ). This mechanism was also supported by analysis of the corresponding rearrangement ions in the MS 2 spectra of the epoxy alcohol formed from 18:3 n -3, 20:2 n -6, and 20:3 n -3 described subsequently. The precise fragmentation mechanism remains to be determined.
MGG_10859 oxidized [8 R -2 H]18:2 n -6 at C-10 and at C-8, and this occurred with retention of the pro-R deuterium label as shown in Fig. 2E . The retention of the deuterium label suggested that the hydroperoxides were formed by antarafacial hydrogen abstraction and oxygen insertion in analogy with hydroperoxides formed by 7,8-LDS of G. graminis and by 10 R -DOX and 5,8-LDS of Aspergillus fumigatus ( 11,13 ).

LC/MS analysis of the epoxy alcohols
The MS 2 spectra of the epoxy alcohols formed from 18:2 n -6 and 18:3 n -3 are shown in Fig. 3 . Both spectra contain unexpected rearrangement ions.  resulting molecular anions confi rmed opening of an epoxide with incorporation of 35 Cl and 37 Cl and formation of a hydroxyl group ( Fig. 3C ). The MS 2 spectrum of the chlorohydrin derivatives showed an intense signal at m/z 311 [A Ϫ -(36 or 38), loss of HCl], and the MS 3 spectrum ( m/z 347-349 → 311 → full scan) yielded the same mass spectrum as recorded for the original epoxy alcohol ( Fig. 3A ). We fi nally confi rmed the structure of the epoxy alcohol by comparison with chemical standards.
Steric analysis of 12(13)-epoxy-10-hydroxy-8(E)-octadecenoic acid. The epoxy alcohol formed from 10 R -HPODE was identifi ed as the syn stereoisomer by its retention time on NP-HPLC in comparison with synthetic and biological standards. Photooxidation of (+)-and ( Ϫ )-vernolic acids, 12 S (13 R )-epoxy-and 12 R (13 S )-epoxy-9( Z )-octadecenoic acids, respectively, yielded two pairs of syn and anti stereoisomers with the same MS 2 spectrum as discussed previously ( Fig. 3A ). The syn and anti stereoisomers were separated by NP-HPLC, but the two syn and the two anti stereoisomers were mirror images, and they were not separated by NP-HPLC.

12(13)-Epoxy-10-hydroxy-8(E),15(Z)-octadecadienoic acid.
The LC/MS spectrum of the epoxy alcohol formed from 18:3 n -3 is shown in Fig. 3E . The presence of an alcohol at C-10 was evident from the strong fragments at m/z 183 and 155 formed by ␣ -cleavage at both sides of C-10. The rearrangement ion at m/z 209 (A Ϫ -100) was apparently formed by the same mechanism as the m/z 211 ion discussed previously. Importantly, this spectrum shows that the rearrangement cannot be formed by cleavage at C-11, which would yield a loss of 98 (OCH-CH 2 -CH = CH-CH 2 -CH 3 ) and not a loss of 100. 18:1n-9. NP-HPLC/MS 2 analysis showed that oleic acid was oxidized by the 10 R -DOX activity to 8-H(P)OME and 10-H(P)OME in a ratio of >5:1. CP-HPLC analysis showed that 8-HPOME consisted of the 8 R stereoisomer to >95%.
18:3n-6. This fatty acid was a poor substrate, and it was not oxidized to specifi c products. The complex spectra of the epoxy alcohol discussed previously did not clearly show the presence of an epoxide. We therefore prepared chlorohydrin adducts, and they were partly separated by RP-HPLC ( Fig. 3B ). The 10-HPODE was altered from 92% in the wild-type enzyme to 29% in the Asn965Val mutant. Asn 965 thus supports the EAS activity, but it is not absolutely required. This is also the case with the catalytically important Asn residues of CYP74 and 9 R -(DOX)-AOS of A. terreus ( 17,37 ); the parentheses indicate that the latter appears to lack 9 R -DOX activity.

Recombinant expression of 7,8-LDS of M. oryzae
We examined recombinant 7,8-LDS for biosynthesis of major and minor metabolites, which might contribute to the oxylipin profi le of DA-99. The recombinant protein transformed 18:2 n -6 sequentially to 8-HPODE and 7,8-Di-HODE as major products. This enzyme was therefore confi rmed to possess 7,8-LDS activities, as previously indicated by gene deletion ( 29 ). NP-HPLC/MS 2 analysis also revealed signifi cant biosynthesis of several minor products as illustrated in Fig. 5B . Trace amounts of 10-H(P)ODE were also formed, but 5,8-DiHODE could not be detected.
The protein sequence of 7,8-LDS contains several predicted phosphorylation sites with Ser and Thr residues ( 38 ). Single residues of fi ve of these sites were altered by replacements with Asp or Glu residues (Ser350Asp in the 8 R -DOX domain, and Ser753Glu, Ser975Asp, Thr998Glu, and Thr1098Asp in the CYP domain), but this did not seem to affect the enzymatic oxidation of 18:2 n -6 (data not shown).
Site-directed mutagenesis studies revealed that Asn 946 , which is present in the AsnGlnXaaGln motif mentioned previously ( Fig. 4A ) 20:2n-6. The 20:2 n -6 was oxidized at C-10 and C-12 as judged from NP-HPLC/MS analysis of hydroperoxides before and after reduction to alcohols. Both hydroperoxides were also converted to epoxy alcohols. The MS 2 spectrum of one epoxy alcohol was consistent with 10(11)-epoxy-12-hydroxy-14( Z )-eicosenoic acid (data not shown), possibly formed by homolytic cleavage of the 10-hydroperoxy metabolite. The MS 2 spectrum of the other epoxy alcohol, 14(15)-epoxy-12-hydroxy-10( E )-eicosenoic acid, is shown in supplementary Fig. IVA, and it allows a comparison with the spectrum in Fig. 3A . This illustrates that many fragments differed by 28, which supports the rearrangement mechanism discussed previously with loss of C 6 H 10 and water from the carboxylate anion.

Conversion of other hydroperoxides by the EAS activity
The capability to convert hydroperoxides other than 10 R -HPODE was evaluated by incubations with 10 S -, 9 R -, 9 S -, 13 R -, and 13 S -HPODE, respectively. Only 13 R -and 13 S -HPODE were converted to epoxy alcohols in significant amounts. Both formed the threo stereoisomer of 12(13)-epoxy-11-hydroxy-9( Z )-octadecenoic acids as the main metabolite ( erythro/threo , 1:4) as judged by NP-HPLC analysis (supplementary Fig. V). Because the hydroperoxides were mostly converted to the threo isomers, it is possible that this occurred enzymatically.

Crucial amino acid residues of the EAS domain of MGG_10859
Catalytically important amino acid residues of the EAS domains are likely conserved residues of the I-helices and the heme thiolate pocket, as outlined by partial alignment of the CYP domains of 10 R -DOX-EAS with three putative DOX-EAS and two 7,8-LDS sequences ( Fig. 4A ).
The Cys1086Ser replacement of MGG_10859 fully supported the oxidation of 18:2 n -6 by the 10 R -DOX activities, but only traces of the epoxy alcohol were noted in comparison with native 10 R -DOX-EAS ( Fig. 4B, C ). These small amounts were presumably formed by the heme group of the 10 R -DOX domain in analogy with 10 R -DOX of A. fumigatus and Aspergillus nidulans , which lack the heme thiolate ligand ( 14,15 ).
We next examined two amide residues in the conserved Asn 965 GlnXaaGln motif of 10 R -DOX-EAS, which is present in three homologs of 10 R -DOX-EAS and in two 7,8-LDS sequences ( Fig. 4A ). The Gln968Leu replacement of 10 R -DOX-EAS did not appear to alter the biosynthesis of the epoxy alcohol from 18:2 n -6, but it was strongly infl uenced by the Asn965Val replacement ( Fig. 4D ). It is diffi cult to exactly quantify this reduction in formation of the epoxy alcohol, but the relative amount of epoxy alcohol to P450) catalyze homolytic cleavage with formation of epoxy alcohols ( 36,39,40 ). 10 R -DOX-EAS likely forms epoxy alcohols by heterolytic cleavage of the O-O bond of 10 R -HPODE with formation of a hydroxyl anion and P450 compound I for subsequent epoxidation ( Fig. 7 ). To the best of our knowledge, 10 R -DOX-EAS is the fi rst described EAS with an intramolecular and position-specifi c oxygenation mechanism associated with heterolytic scission of the dioxygen bond. Heterolytic scission of dioxygen bonds are also catalyzed by heme peroxidases and presumably also by the secreted EAS of Magnaporthe salvinii ( 41,42 ). Intramolecular oxygen transfer has also been described by arachidonate 12-Lipoxygenase linked to diol synthase activities in the red alga Gracilariopsis lemaneiformis and by 15-Lipoxygenase linked to epoxygenase activities of the fungus Saprolegnia parasitica ( 1,43 ).
10 R -DOX-EAS contains the conserved Asn 965 GlnXaaGln motif, presumably located in the I-helix of the EAS domain ( Fig. 4A ). The Asn residue of this sequence supports catalysis of both 7,8-LDS of G. graminis and M. oryzae (cf. Fig. 6 and supplementary Fig. VII) ( 9 ). In analogy, 10 R -DOX-EAS•N965V restrained the conversion of 10 R -HPODE to 12 S (13 R )-epoxy-10( R )-hydroxy-8( E )-octadecenoic acid ( Fig. 3 ). It is therefore possible that Asn 965 is involved in positioning of 10 R -HPODE for heterolytic scission of the oxygen-oxygen bond in analogy with the homologous Asn residues of 7,8-LDS. The position of the heme iron in the active sites appears to differ in relation to the hydroperoxides at C-8 and C-10. 7,8-LDS catalyzes hydroxylation at C-7 of 8 R -HPODE, whereas 10 R -DOX-EAS catalyzes epoxidation of the 12( Z ) double bond of 10 R -HPODE. Future work will

DISCUSSION
The discovery of the fi rst fungal EAS with its unique selfsuffi cient reaction mechanism is our main fi nding. The dual enzyme activities are combined in a DOX-CYP fusion protein, MGG_10859, of the rice blast fungus M. oryzae . The enzyme is designated 10 R -DOX-EAS because it sequentially converts linoleic and ␣ -linolenic acids to 10 R -hydroperoxides and to 12 S (13 R )-epoxy-10 R -hydroxy metabolites as end products by intramolecular oxygen transfer ( Fig. 7 ).
Epoxy alcohols can be formed from fatty acid hydroperoxides by two main mechanisms: homolytic or heterolytic cleavage of the hydroperoxide oxygen-oxygen bond. Hematin, hemoproteins, and many enzymes (e.g., lipoxygenases and ( Fig. 4A ). Two sequences with fi ve conserved amino acids are found in P450s with relatively few exceptions, namely Glu and Arg residues in the salt bridge ("EXXR") and Cys and two Gly residues in the heme thiolate pocket ("GXXX-CXG") ( 19 ). The other amino acids in these two sequences are characteristic of fungal P450 families ( 19 ).
The PMK1 and cAMP-activated PKA constitute two important pathways for the infectious process of M. oryzae (25)(26)(27)45 ). The biological function of 10 R -DOX-EAS is unknown, but constitutive PKA expression alters the oxylipin profi le of M. oryzae , and PMK1 affects expression of MGG_10859 mRNA. The latter is upregulated during the early and late phases of appressoria formation and downregulated by gene deletion of PMK1 6 ( 30 ). It raises the question of whether 10 R -DOX-EAS also could be regulated by cAMP-activated PKA.
The strain DA-99 of M. oryzae with the suppressor mutant ⌬ mac1 sum1-99 ( 23 ) has constitutive PKA activities. Nitrogen powder of this fungus formed 8 R -HPODE and 10 R -HPODE as main metabolites, but without signifi cant further transformation by EAS and only with modest biosynthesis of 7,8-DiHODE in comparison with M. oryzae Guy11 ( Fig. 5 ). There are no other known gene candidates for these 8 R and 10 R activities than 10 R -DOX-EAS and 7,8-LDS of M. oryzae . The altered oxylipin profi le is therefore likely related to these enzymes, either by direct effects of PKA on these proteins or by indirect effects on their gene expression. reveal whether 8 R -and 10 R -HPODE bind the active sites with their conserved residues in opposite directions.
10 R -DOX-EAS of M. oryzae is the fi rst member of a distinct subfamily of DOX-CYP fusion proteins ( Fig. 1B ). The amino acid identity of this enzyme with 10 R -DOX of A. fumigatus and A. nidulans with nonfunctional CYP domains is ‫ف‬ 47% over the entire sequence and only slightly higher ( ‫ف‬ 51%) over the 10 R -DOX domains ( ‫ف‬ 680 N-terminal amino acids), whereas these two aspergilli enzymes can be aligned with 78% amino acid identity (cf. Fig. 1B ). The n -6 double bond is essential for oxygenation at C-10, as linoleate 10 R -DOX-EAS transforms 18:1 n -9 to 8-HPOME as main metabolite in analogy with 10 R -DOX of aspergilli ( 14,15 ). Alignments of 10 R -DOX and 10 R -DOX-EAS sequences clearly indicate two distinct subfamilies, and 10 R -DOX does not appear to be an inactive form of 10 R -DOX-EAS ( Fig.  1B and supplementary Fig. VIII). The fact that two 10 R -HPODE-producing subfamilies have evolved in parallel strengthens previous reports that 10 R -HPODE is of biological importance ( 44 ).
A second 10 R -DOX-EAS (FOXB_03425 7 ) was identifi ed in F. oxysporum by recombinant expression. Sequence homologs are present in at least two other devastating fungal pathogens ( Fig. 1B ), and it seems likely that the latter also possess 10 R -DOX-EAS activities. Virtually identical are the sequences of the homologs around the proximal and distal heme ligands of the10 R -DOX domains (not shown) and critical regions (I-helices) of the EAS domains  Phosphorylation, in particular by PKA, has previously been found to downregulate the catalytic activity as a functional switch of several isoforms of the CYP2 family ( 46 ). 7,8-LDS and 10 R -DOX-EAS contain several phosphorylation sites. We hypothesized that phosphorylation of 7,8-LDS might alter the catalysis. However, replacement of Ser and Thr with Asp or Glu residues in fi ve predicted phosphorylation sites did not change the oxylipin profile (cf. Ref 47 ). PKA influences gene expression by phosphorylation of transcription factors and by alternative splicing of pre-mRNA ( 48 ). A. terreus forms a splice variant of 5,8-LDS with retention of the last intron, leading to a premature stop codon with complete loss of the P450 catalysis ( 17 ). Alternative splicing of 7,8-LDS and 10 R -DOX-EAS in this way could lead to accumulation of 8 R -and 10 R -HPODE, and this hypothesis merits further investigation.

CONCLUSION
We have discovered and characterized catalytic properties of the fi rst 10 R -DOX-EAS. This fusion enzyme catalyzes "oxygen transfer" epoxidation and belongs to a novel DOX-CYP subfamily with homologs in several of the top 10 fungal plant pathogens ( 20 ). Previous work suggests that the underlying gene, MGG_10859, could be regulated by the gene PMK1 , which is a key regulator of the infectious process ( 30 ). Based on these results, the biological function of 10 R -DOX-EAS and its enzymatic products can now be assessed. Fig. 7. Enzymatic conversion of 18:2 n -6 by recombinant 10 R -DOX-EAS. Abstraction of the pro-S hydrogen of C-8 is followed by antarafacial oxygen insertion mainly at C-10 and to some extent at C-8, yielding 10 R -and 8 R -HPODE, respectively. 10 R -HPODE is further converted by the EAS activity to 12 S (13 R )-epoxy-10( R )-hydroxy-8( E )-octadecenoic acid by heterolytic scission of the O-O bond with formation of P450 compound I in a process supported by Asn 965 (cf. Fig. 4 ).