Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum.

Fusarium oxysporum is a devastating plant pathogen that oxidizes C18 fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C18 and C20 fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu946-Val-Leu-Ser949, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.

the dioxygen bonds of 8 R -HPODE, and Asn 964 of the AOS facilitates homolytic cleavage of the dioxygen bond of 9 R -HPODE ( 18,25 ). The ClustalW alignment suggests that Asn 964 and Gln 967 of the AOS of A. terreus are replaced by an acidic and an alcoholic residue, Glu 946 and Ser 949 respectively , in FOXB_01332 ( Fig. 1 ). The latter sequence also contains two amide residues, Asn 921 and Gln 924 , not far from the homologous position of Asn 964 of ATEG_02036 (supplementary Fig. I).
The fi rst objective of the present investigation was to express FOXB_01332 and to study its catalytic activity. Our experiments demonstrate that FOXB_01332 is a 9 S -DOX-AOS fusion protein. The 9 S -DOX motif, Tyr-Arg-Phe-His, occurs in a subset of fungal DOX-CYP fusion proteins ( Fig. 1 ). The second objective was to determine whether the Phe residue in this sequence was essential for 9 S -DOX activity. The third objective was to compare potentially important residues of the two fungal AOSs.
The F. oxysporum species complex contains a wide range of plant pathogens and producers of a plethora of mycotoxins, and it comprises two of the top 10 fungal pathogens, F. oxysporum and Fusarium graminearum ( 19 ). Their genomes have been sequenced to facilitate research on their pathogenic mechanism and this information is available at the Fusarium Comparative Database . We hypothesized that F. oxysporum might produce allene oxides because its genome encodes homologous enzymes to the AOS of A. terreus . FOXB_01332 could be aligned to this AOS with high amino acid identity ( ‫ف‬ 50%, see below ) (supplementary Fig. I).
The AOS of A. terreus belongs to the family of fungal DOX-CYP fusion proteins. The latter contain linoleate 8 R -DOX or 10 R -DOX in their N-terminal domains and 5,8-or 7,8-hydroperoxide isomerases or an AOS in the C-terminal CYP domains ( 18,(20)(21)(22)(23); 9 R -DOX activity could not be detected in the recombinant AOS of A. terreus . This fusion protein thus differs from the AOS of the coral Plexaura homomalla , which contains an N-terminal catalase-like domain with AOS activity and a C-terminal 8 R -LOX domain ( 24 ).
The DOX domains of DOX-CYP fusion enzymes are homologous to COX, as illustrated in Fig. 1 by a comparison of the tetramer sequence including the tyrosine residue which catalyzes hydrogen abstraction and the proximal histidine heme ligand. These tetrameric sequences of ATEG_02036 and FOXB_01332 are identical, but differ at two positions from COX and at one from 5,8-and 7,8linoleate diol synthase (LDS) with 8 R -DOX activities ( Fig. 1 ). Whether the evolutionary replacement of Trp with Phe in ATEG_02036 and FOXB_01332 supports DOX activities is unknown.
Comparison of the C-terminal sequences of ATEG_02036 and FOXB_01332 revealed an interesting difference. The Asn and Gln residues in the pentamer sequence (Ala-Asn-Xaa-Xaa-Gln) of 5,8-LDS and the AOS of A. terreus are of catalytic importance ( 18 ). These amide residues of 7,8-LDS and 5,8-LDS support the heterolytic cleavage of  mass spectra were recorded every second and consisted of an average of fi ve microscans, each with a maximum injection time of 0.2 s. The mass spectra were routinely analyzed with selective ion monitoring of characteristic ions and also checked by subtraction of background spectra before and after the peaks of interest. These chromatograms were analyzed with the Xcalibur software with peak integration and manual selection of the integration set points.

Bioinformatics
Proteins were aligned with the ClustalW algorithm (Lasergene, DNASTAR, Inc.). Phylogenetic trees were constructed with the MEGA5 software with bootstrap tests of the resulting nodes ( 29,30 ). The distance within branches was based on the number of expected substitutions per amino acid position.

Bioinformatics
The genomes of the F. oxysporum complex, which are sequenced at the Broad Institute, were investigated for homologs of the AOS of A. terreus . FOXB_01322 of F. oxysporum Fo5176 (GenBank, EGU88194) could be aligned with ‫ف‬ 50% amino acid identity with the AOS of A. terreus (Gen-Bank, AGH14485 or ATEG_02036). An alignment of the two sequences is shown in supplementary Fig. I. As expected, the NCBI database identifi ed LDS-and CYP-like domains in FOXB_01332. We therefore decided to investigate FOXB_01322 for DOX and AOS activities.
(>98%), and [11 R -2 H]18:2n-6 (25%) were generous gifts of Dr. M. Hamberg (Karolinska Institute, Stockholm). The Champion pET Directional TOPO kit was from Invitrogen. Restriction enzymes and chemically competent Escherichia coli (NEB5 ␣ ) were from New England BioLabs, Fermentas, and Invitrogen. The plasmid Midi kit was from Macherey-Nagel and the gel extraction kit and Pfu DNA polymerase were from Fermentas. Phusion DNA polymerase was from New England BioLabs. RNaseA and ampicillin were from Sigma. Primers were obtained from TIB MOL-BIOL (Berlin, Germany). Sequencing was performed at Uppsala Genome Center (Rudbeck Laboratories, Uppsala University).

Expression in E. coli
The open reading frame of FOXB_01332 (GenBank, EGU88194) was purchased in pUC57 from GenScript (Hong Kong) and was subcloned to pET101D-TOPO vectors by PCR technology (forward primer: 5 ′ -caccatgtcgttcaatgaaaag, reverse primer: 5 ′ -atttcccaaaatctcatcctttc) following Invitrogen's instructions. The amplicon was ligated into pET101/D-TOPO and introduced into E. coli (BL21) by heat-shock transformation. Cells were grown to A 600 0.6-0.8 in low salt Luria Bertani medium and protein expression was induced by 0.1 mM isopropyl ␤ -D-1-thiogalactopyranoside. Cultures were grown for 5 h at room temperature with moderate shaking ( ‫ف‬ 100 rpm). Cells were harvested by centrifugation and sonicated (Bioruptor Next Gen, 10 × 30 s, 4°C) ( 23 ). At least three independent expressions were analyzed.

Site-directed mutagenesis
Site-directed mutagenesis was performed according to the Quick Change protocol (Stratagene) in pUC57 constructs ( 23 ). Amplicons were obtained from 10 ng of template by Pfu DNA polymerase (16 cycles) before digestion with Dpn1 (2 h, 37°C). Amplifi cation of one distinct PCR product was confi rmed by agarose gel electrophoresis before heat-shock transformation (NEB5 ␣ ). Primers containing the designated replacements are listed in the supplementary material (supplementary Table I). All mutations were confi rmed by sequencing before subcloning to pET101D-TOPO as described above.

LC-MS/MS analysis of oxylipins
Reversed phase (RP)-HPLC with MS/MS analysis was performed with a Surveyor MS pump (Thermo Fisher) and an octadecyl silica column (5 m, 2.1 × 150 mm; Phenomenex), which was usually eluted at 0.3-0.5 ml/min with methanol/water/acetic acid, 800/200/0.05 or 750/250/0.05. The effl uent was subject to electrospray ionization in a linear ion trap mass spectrometer (LTQ, Thermo Fisher) with monitoring of carboxylate anions. The heated transfer capillary was set at 315°C; the ion isolation width at 1.5, 4, or 5 amu; the collision energy at 35 (arbitrary scale); and the tube lens varied between 90 and 120 V. PGF 1 ␣ was infused for tuning. The This is in agreement with antarafacial hydrogen abstraction and oxygenation.

Product profi le over time and chromatographic reproducibility.
Analysis of the total ion current during RP-HPLC-MS/MS analysis of metabolites formed from 18:2n-6 by 9 S -DOX-AOS showed that 9-HODE was the main product. The ion current averaged 67% at short incubation time (1 min), whereas the ␣ -ketol was the main product after 3 min (63%) and dominated at later time points (10 min, 87%; 30 min, 91%), as illustrated in Fig. 4 . The corresponding numbers of the total ion current of a second experiment were 73% 9-HPODE at 1 min, and 61, 89, and 94% ␣ -ketol at the later time points. Ten replicate analyses of the same As outlined in Fig. 1 and the introduction, the short sequence with the proximal heme ligand and the important Tyr residue for 8 R -and 10 R -DOX catalysis were identical in FOXB_01332 and ATEG_02036. All ascomycetous sequences with over 50% identity to FOXB_01332/ ATEG_02036 contained a Trp instead of the Phe in the Tyr-Arg-Phe-His motif of the DOX domains.
FOXB_01332 and ATEG_02036 differed in an interesting way in the CYP domain as mentioned above. The AOS and 5,8-LDS of A. terreus contain the conserved sequence Ala-Asn-Xaa-Xaa-Gln, and the amide residues, Asn and Gln, are important for catalysis ( 18 ). At the homologous positions of FOXB_01332, Asn 964 and Gln 967 are replaced with an acidic (Glu 946 ) and an alcoholic residue (Ser 949 ). There are several Asn residues in the putative I-helix of FOXB_01332, and one of them is present within a similar tetramer, Asn 921 -Val-Asp-Gln (supplementary Fig. I).

Oxidation of ␣ -linolenic linoleic acids
Biosynthesis of 9S-hydroperoxides. FOXB_01332 was expressed in E. coli and was found to possess both 9 S -DOX and AOS activities and will therefore be referred to as 9 S -DOX-AOS.
The 18:2n-6 was oxidized by 9 S -DOX-AOS in the same way as 18:3n-3. Steric analysis by CP-HPLC-MS/MS showed that 95% 9 S -HODE and 5% 9 R -HODE were formed ( Fig. 3A ). Small amounts of the trans isomer of 9-HODE were also detected. The relatively low stereospecifi city of 9 S -DOX compared with 8 R -and 10 R -DOX is likely due to rapid transformation of 9 S -HPODE by the AOS, which leads to the accumulation of autoxidation products.

Oxidation of other fatty acids
The 18:3n-6 was oxidized at C-9, but this hydroperoxide was not transformed by the AOS activity ( Fig. 7A ). The 18:4n-3 was oxidized by hydrogen abstraction at C-11 and C-8 with oxygen insertion mainly at C-9, C-10, and C-11 ( Fig. 7B ). The 9-hydroperoxide of 18:4n-3 was partly transformed by the AOS activity with hydrolysis to an ␣ -ketol as judged from the MS/MS and MS 3 spectra with similar fragmentation to the ␣ -ketols described above.
We next examined the effect of chain elongation to C 20 and shortening to C 16 . The 20:2n-6 and 20:3n-6 were oxidized at C-11, and these hydroperoxides were almost sample by RP-HPLC-MS/MS showed that the product profi le was reproducible, as the relative amount of the ␣ -ketol in this sample averaged 72.4% with a standard deviation of 1.55%.

D-KIE of 9S-DOX-AOS.
The D-KIE during the sequential oxidation to 9-HPODE and ␣ -ketols was estimated by comparison of the rate of oxidation of [11,11-2 H 2 ]18:2n-6 and [ 13 C 18 ]18:2n-6 as illustrated in Fig. 5B . The D-KIE for formation of ␣ -ketols ranged between 3 and 5 , suggesting little or no hydrogen tunneling in the sequential biosynthesis of the 9-hydroperoxide and the allene oxide/ ␣ketol. Analysis of the formation rate of the 9-hydroperoxide from deuterated and 13 C-labeled 18:2n-6 yielded similar results. We conclude that 9 S -DOX catalyzes hydrogen abstraction with a small D-KIE in analogy with other DOX of the animal peroxidase gene family.

Transformation of hydroperoxides by AOS
9 S -DOX-AOS converted 9 S -HPODE to ␣ -and ␥ -ketols, likely formed by hydrolysis of 9 S (10)-EODE as discussed above, along with small amounts of epoxy alcohols ( Fig. 6A ). The 9 R -HPODE preparation contains a few percent 9 S -HPODE, and only the latter was apparently transformed to an ␣ -ketol by 9 S -DOX-AOS ( Fig. 6B ). 13 S -HPODE, 13 S -HPOTrE, 13 R -HPODE, and 13 R -HPOTrE were not transformed to detectable amounts of allene oxides/ ␣ -ketols, but variable amounts of epoxy alcohols accumulated ( Fig. 6 ).  S -DOX activity abstracts the pro R hydrogen at C-11, whereas the ␣ -ketol may lose the remaining deuterium label at C-11 due to keto-enol tautomerism. The D-KIE was therefore estimated by MS 3 with a mass window of four during the fi rst and second selection, as indicated in the fi gure. 9-HPODE with formation of the ␣ -ketol as the main product (>90%), but an increased formation of other hydroperoxides could not be detected. We conclude that the replacement of Phe 416 with Trp does not affect the product profi le of FOXB_01332.
Site-directed mutagenesis of the AOS. The LDS and AOS of A. terreus contain the conserved sequence Ala-Asn-Xaa-Xaa-Gln in their CYP domains ( Fig. 1 ). These Asn and Gln residues were shown to be crucial for the hydroperoxide isomerase activities of 5,8-and 7,8-LDS, respectively, and the Asn residue for the AOS activity ( 18,25 ). At the homologs positions according to ClustalW alignments, 9 S -DOX-AOS contains an acidic and an alcoholic residue (Glu 946 and Ser 949 , respectively) ( Fig. 1 ). In order to determine whether these residues were critical for the AOS activity, we replaced them with Val and Ala residues, respectively. The mutant Glu946Val oxidized 18:2n-6 as the completely further transformed by the AOS activity followed by nonenzymatic hydrolysis to ␣ -ketols (supplementary The 16:3n-3 was a poor substrate and was mainly oxidized by hydrogen abstraction at C-12 with formation of 10-hydro(pero)xy-7( Z ),11( E ),13( Z )-hexadecatrienoic acid without further transformation by the AOS activity (data not shown). This metabolite is also formed by L. theobromae ( 34 ).
We conclude that the DOX activity appears to have a relatively broad substrate specifi city for unsaturated C 18 to C 20 fatty acids, whereas the AOS activity appears to be specifi c for 9 S -and 11 S -hydroperoxides of C 18 and C 20 fatty acids with the notable exception of 9 S -HPOTrE(n-6) ( Fig. 7A ).

Site-directed mutagenesis of 9 S -DOX-AOS
Site-directed mutagenesis of 9S-DOX. The conserved Tyr-(His/Arg)-Trp-His motif contains the catalytic Tyr residue, which is oxidized by the heme to a radical that performs the hydrogen abstraction of LDS and COX ( Fig. 1 ). ATEG_02036 and FOXB_01332 retain the Tyr residue but have a Phe residue instead of a Trp in this sequence. To assess whether this residue could be of catalytic importance, we replaced Phe 416 of FOXB_01332 with a Trp residue. The Phe416Trp mutant oxidized 18:2n-6 to Fig. 7. LC-MS analysis of the oxidation of 18:3n-6, 18:4n-3, and 18:1n-6 by 9 S -DOX-AOS. A: The 18:3n-6 was transformed to the 9-hydroperoxide (9-HPOTrE) as the main product and to traces of epoxy alcohols. B: The 18:4n-3 was oxidized mainly at C-9, C-10, and C-11, and relatively small amounts of ␣ -ketol were also detected (data not shown). C: The 18:1n-6 was oxidized at C-11 and resolved by CP-HPLC to one major isomer and this hydroperoxide was likely the 11 R stereoisomer of 11-HPOME; oxygen is presumably inserted at C-11 from the same direction as in the biosynthesis of 9 S -HPODE, but designated 11 R-HPOME due to the Cahn-Ingold-Prelog priority rules. The metabolites were separated by normal phase HPLC. TIC, total ion current. enzymes, which now consists of LDS, 10 R -DOX, AOS, and 9 S -DOX-AOS. The two AOSs form different allene oxides, 9 S (10)-EODE and 9 R (10)-epoxy-11,(12 Z )-octadecadienoic acid, and they are not homologous to plant AOSs of the CYP74 family ( 35 ). The two AOSs also differ in the active site as the position of the catalytically important Asn 941 residue for homolytic cleavage of 9 R -HPODE is not conserved in the homologous position of 9 S -DOX-AOS ( 18 ). The fact that F. oxysporum has the capacity to form allene oxides from 9 S -HPODE in analogy with plants suggests that these allene oxides may have biological functions, but little is yet known about the biological role of AOS other than in JA biosynthesis ( 8 ), and whether additional AOSs occur in pathogens of the Fusarium complex.

S -DOX of F. oxysporum and R -DOX of A. terreus
The 9 S -DOX activity of FOXB_01332 with 18:2n-6 and 18:3n-3 as substrates shows many similarities to 9 S -LOX of plants, and it may therefore be mistaken as a LOX. 9 S -DOX and 9-LOX catalyze antarafacial hydrogen abstraction and oxygenation ( 27,37 ). A detailed analysis revealed two characteristic differences between hydrogen abstraction by a tyrosyl radical and by a LOX metal center with a catalytic base, e.g., Fe 3+ OH Ϫ . First, the D-KIE of 9 S -DOX-AOS was in the same order of magnitude as reported for COX ( 38 ), and at least one order of magnitude less than the D-KIE of sLOX -1 ( 39 ). Second, 9 S -DOX oxidizes 18:1n-6 by hydrogen abstraction at C-11 in analogy with the effi cient oxidation of 18:1n-9 at C-8 by 8 R -DOX of native enzyme with formation of the ␣ -ketol as the main product after 30 min, as judged from the total ion current (range 87-93%; n = 3). The Ser949Ala mutant also formed ␣ -ketol as the native enzyme. This acid-alcohol pair thus lacks catalytic importance for the AOS activity.
The putative I-helix of the AOS domain contains several Asn residues, one of which is present in the sequence Asn 921 -Val-Asp-Gln (supplementary Fig. I). Asn residues appear to be critical for all AOSs identifi ed so far ( 18,20,35,36 ). We therefore examined whether replacement of Asn 921 with Val could infl uence the AOS activity of FOXB_01332 in analogy with Asn 964 of AOS of A. terreus . The Asn921Val mutant oxidized 18:2n-6 with formation of 9-HPODE, ␣ -ketols, and ␥ -ketols as the native 9S-DOX-AOS (data not shown).

DISCUSSION
We have expressed the fi rst 9 S -DOX, and it belongs to the animal heme peroxidase gene family. This enzyme was found to be fused with a CYP domain, which encodes an AOS. Our report demonstrates a novel route to allene oxides without the need of a LOX. An overview of the catalytic domains and the reaction mechanism of 9 S -DOX-AOS are shown in Fig. 8A .

Biological relevance
The discovery of the 9 S -DOX-AOS has biological implications. It extends the family of fungal DOX-CYP fusion . The latter desaturates the 9(10)-epoxide intermediate with a radical at C-11 and forms the double bond between C-10 and C-11. B: Nonenzymatic hydrolysis of 9 S (10)-EODE yields ␣ketols and small amounts of ␥ -ketols. The relative amounts of the two ␣ -ketols differ during nonenzymatic hydrolysis of 9 S (10)-EODE due to steric hindrance at C-9.
The acid-alcohol pair in the sequence Gly-Xaa-(Glu/ Asp)-(Ser/Thr) of CYP hydroxylases participates in cleavage of molecular oxygen ( 45 ). The acidic (Glu 946 ) and alcoholic (Ser 949 ) residues of FOXB_01332 at the homologous position of the amide residues in Ala-Asn-Xaa-Xaa-Gln of LDS and AOS of A. terreus appeared to lack catalytic importance (cf. Fig. 1 ). Replacement of these Glu and Ser residues with nonpolar amino acids did not alter the AOS activity.
Asn residues facilitate the AOS activities of plant and fungal AOS ( 18,20,35,36 ). The putative I-helix of 9 S -DOX-AOS contains four Asn and four Glu residues (supplementary Fig. I). One of them, Asn 921 , is present in the sequence Asn-Asn 921 -Val-Asp-Gln. Replacement of Asn 921 with Val did not alter the AOS activity. Additional work is needed to determine whether any of the other Asn residues facilitate the homolytic cleavage of 9 S -HPODE.

Homologs to DOX-AOS
Our results raise the question whether the two fi rst steps of fungal JA biosynthesis are catalyzed by separate 13 S -LOX and AOS as in plants or by putative DOX-AOS fusion proteins. Sequence analysis of ascomycetous fungi revealed several proteins with at least 50% amino acid identity to 9 S -DOX-AOS and AOS of A. terreus ( Fig. 9 ). All these proteins contained the Tyr-Arg-Phe-His motif in the DOX domains and several consensus sequences of animal heme peroxidases, whereas the CYP domains contained relatively few conserved sequences in addition to the Glu-Xaa-Xaa-Arg salt bridge motif and the catalytic loop with the heme thiolate ligand [Gly-Xaa-His-Glu-Cys-Xaa-(Ala/Gly)].
The two fungal AOS domains also lack characteristic motifs in support of homolytic cleavage of 9-HPODE other than the thiolate heme ligand. It might therefore be difficult to identify novel fungal AOS by bioinformatics alone.
A. terreus and L. theobromae possess prominent 9 R -DOX activities, which are linked to AOS ( 17,34 ). The 9 R -DOX activity of A. terreus retains a broad substrate specifi city and a D-KIE of the same magnitude as 9 S -DOX, but it catalyzes suprafacial hydrogen abstraction and oxygenation ( 17,18 ).
The sequence homology between the DOX domains of FOXB_01332 and ATEG_02036 is intriguing. It raises two questions. Could the lack of 9 R -DOX activity of recombinant ATEG_02036 be due to cloning or expression artifacts? If this is the case, what is the structural basis of supra-or antarafacial oxygen insertion? 9 R -DOX could also be unrelated to ATEG_02036 and 9 S -DOX, but the low D-KIE of 9 R -DOX is consistent with other DOXs of the animal peroxidase gene family ( 38,42 ).

AOS of F. oxysporum and A. terreus
The AOS activity of FOXB_01332 converted 9 S -hydroperoxides of C 18 fatty acids as well as 11 S -hydroperoxides of C 20 fatty acids. Importantly, 9 R -, 13 S -, and 13 R -hydroperoxides were not transformed. It is therefore unlikely that this AOS is involved in the biosynthesis of allene oxides as precursors of JA. 9 S -HPODE undergoes homolytic cleavage by the AOS. This likely occurs with formation of CYP[Fe IV OH] and an intermediate, 9 S (10 R )-epoxy-12( Z )-octadecenoic acid, with a radical at C-11, followed by biosynthesis of an allene oxide by electron transfer from C-11 to the metal center and proton loss, as described for plant AOS ( 35,43 ). Allene oxides are unstable in aqueous solutions with nonenzymatic hydrolysis to ␣ -and ␥ -ketols as illustrated in Fig.  8B , but allene oxides can be isolated and purifi ed at low temperatures ( 7,44 ).
We expected the catalytically important Asn 964 of AOS of A. terreus to align with an important region of 9 S -DOX-AOS. Fig. 9. Phylogenetic tree of protein sequences of ascomycetes with at least 50% sequence identity to AOS of A. terreus and/or 9 S -DOX-AOS of F. oxysporum . The phylogenetic tree (MEGA5) is based on the alignment of selected protein sequences from the ten listed organisms and, for reference, 7,8-LDS of G. graminis and 5,8-LDS of A. terreus (marked in blue). In addition to LDS, the only nonorphan proteins are 9 S -DOX-AOS of F. oxysporum and AOS of A. terreus (marked in red). The horizontal bar indicates an estimation of the substitution rate per amino acid position ( 29 ). The GenBank ID numbers of the proteins are: EFQ27323, Colletotrichum graminicola ; CCF39565, Colletotrichum higgensianum ; ENH82400, Colletotrichum orbiculare ; EKJ79444, Nevertheless, synthesized open reading frames of candidate genes can be used for protein expression. This may provide important information on DOX-CYP proteins and JA biosynthesis by the F. oxysporum complex. CONCLUSIONS A 9 S -DOX and an AOS are present as a fusion protein of F. oxysporum. 9 S -DOX is homologous to LDS, 10 R -DOX, and other animal heme peroxidases, and the AOS is a CYP with homology to the AOS of A. terreus . The homolytic cleavage of 9 R -and 9 S -HPODE by the heme thiolate group of these AOSs is facilitated by different amino acids, and these enzymes may therefore belong to separate DOX-AOS subfamilies.