A new class of fatty acid allene oxide formed by the DOX-P450 fusion proteins of human and plant pathogenic fungi, C. immitis and Z. tritici[S]

Linoleate dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are common in pathogenic fungi. The DOX domains form hydroperoxy metabolites of 18:2n-6, which can be transformed by the CYP domains to 1,2- or 1,4-diols, epoxy alcohols, or to allene oxides. We have characterized two novel allene oxide synthases (AOSs), namely, recombinant 8R-DOX-AOS of Coccidioides immitis (causing valley fever) and 8S-DOX-AOS of Zymoseptoria tritici (causing septoria tritici blotch of wheat). The 8R-DOX-AOS oxidized 18:2n-6 sequentially to 8R-hydroperoxy-9Z,12Z-octadecadienoic acid (8R-HPODE) and to an allene oxide, 8R(9)-epoxy-9,12Z-octadecadienoic acid, as judged from the accumulation of the α-ketol, 8S-hydroxy-9-oxo-12Z-octadecenoic acid. The 8S-DOX-AOS of Z. tritici transformed 18:2n-6 sequentially to 8S-HPODE and to an α-ketol, 8R-hydroxy-9-oxo-12Z-octadecenoic acid, likely formed by hydrolysis of 8S(9)-epoxy-9,12Z-octadecadienoic acid. The 8S-DOX-AOS oxidized [8R-2H]18:2n-6 to 8S-HPODE with retention of the 2H-label, suggesting suprafacial hydrogen abstraction and oxygenation in contrast to 8R-DOX-AOS. Both enzymes oxidized 18:1n-9 and 18:3n-3 to α-ketols, but the catalysis of the 8R- and 8S-AOS domains differed. 8R-DOX-AOS transformed 9R-HPODE to epoxy alcohols, but 8S-DOX-AOS converted 9S-HPODE to an α-ketol (9-hydroxy-10-oxo-12Z-octadecenoic acid) and epoxy alcohols in a ratio of ∼1:2. Whereas all fatty acid allene oxides described so far have a conjugated diene impinging on the epoxide, the allene oxides formed by 8-DOX-AOS are unconjugated.

Z. tritici might code for known or unique enzymes with homology to 8R-DOX-LDS and related enzymes.
C. immitis and Z. tritici code for three DOX-CYP fusion enzymes each. These tentative proteins can be aligned with characterized DOX-CYP, as shown by the phylogenetic tree in Fig. 1B. The alignment suggests that EGP91582 and EAS36125 are likely related to 8R-DOX-LDS. EAS34688 is connected to 10R-DOX-(CYP), but EAS34688 retains the heme-thiolate cysteine in the CYP domain in contrast to 10R-DOX-(CYP) and might therefore be catalytically related to the nearby 10R-DOX-EAS subfamily (see Fig. 1B). The catalytic properties of the three remaining enzymes are more difficult to deduce.
EAS28473 and EGP83657 appear to be only distantly related to the known DOX-CYP subfamilies, but they can be aligned with 51% amino acid identity (Fig. 1B). EGP87976 aligns with both 9R-and 9S-DOX-AOS, and could be catalytically related. All three proteins have conserved the proximal and distal His heme ligands and the catalytically important Tyr residue in the DOX domains and the critical heme thiolate cysteine in the CYP domains.
We decided to express and characterize EAS28473, EGP83657, and EGP87976 for three reasons. First, they appeared to form, on sequence alignment with related of 18:2n-6 can also be subject to homolytic cleavage and dehydration to allene oxides by allene oxide synthases (AOSs) (9R-and 9S-DOX-AOS) (Fig. 1A). AOSs of plants belong to the CYP74 family, but the fungal AOSs form separate CYP families (17,18). The 8R-DOX-LDS is often expressed by mycelia in laboratory cultures of many strains, whereas other DOX-CYP may be expressed by certain strains or only in response to specific environmental stimuli (1,19).
The DOX-CYP enzymes can also be classified from the position of hydrogen abstraction of linoleic acid: C-8 by 8R-and 10R-DOX and C-11 by 9-DOX (12,15,17). The general theme is antarafacial hydrogen abstraction and oxygen insertion in analogy with COX, but there are two exceptions: 9R-DOX and 9R-DOX-AOS (17,18,20).
Coccidioides immitis causes valley fever in Western USA with occasionally lethal outcomes (21). Zymoseptoria tritici (teleomorph Mycosphaerella graminicola) causes the most important disease of wheat, septoria tritici blotch (22,23). Little is known about the DOX-CYP enzymes of these important pathogens. Fungal oxylipins may take part as secondary metabolites in sporulation, the infectious processes, and in biotrophic and necrotic growth (1,2). It therefore seemed of interest to determine whether C. immitis and A: Overview of fungal oxylipins formed by DOX-CYP fusion enzymes. Linoleic acid is transformed by mycelia and/or by recombinant enzymes of subfamilies of DOX-CYP fusion to 1,2-and 1,4-diols (left, marked green), to allene oxides (right, marked blue), to an epoxy alcohol (bottom, black), and to 10R-HPODE (top, black). The parentheses in 10R-DOX-(CYP) indicate that the CYP domain is homologous to P450 but lacks the critical Cys residue for catalysis. B: Phylogenetic tree of characterized DOX-CYP fusion enzymes except for three orphans each of C. immitis and Z. tritici (marked red). The sequences are (GenBank identification numbers): EDP52540 and AFB71131 for 10R-DOX-(CYP); EGU86021 and EFQ36272 for 10R-DOX-EAS; for three subfamilies of 8R-DOX-LDS: AGA95448 and EDP50447 for 5,8-LDS, EHA52010 and Q9UUS2 for 7,8-LDS, KDN68726 and EFQ34869 for 7,8-and 8,11-LDS; EGU88194 and EFQ27323 for 9S-DOX-AOS; AGH14485 and EHA29500 for 9R-DOX-AOS; EGU79548 and EFQ36675 for 9R-DOX. experiments, DNase I (about 0.1 mg/ml). The suspension was frozen and thawed twice and then sonicated (Bioruptor Next Gen; 10 times (30 s sonication, 30 s pause), 4°C). Cell debris was removed by centrifugation and the supernatants were used immediately or stored at 80°C until needed. EAS28473, EGP83657, and EGP87976 were expressed in more than three independent experiments.
We confirmed that cell lysate of untransformed E. coli BL21 Star cells does not oxidize 18:2n-6 to any of the products discussed below. Analysis of cell lysates of transformed E. coli without added 18:2n-6 did not form any metabolites of this acid.

Enzyme assays
Recombinant proteins of the crude cell lysate [in 0.05 M Tris-HCl (pH 7.6)/5 mM EDTA/10% glycerol or 0.05 M KHPO 4 (pH 7.5)/0.3 M NaCl/0.1 M KCl/1 mM GSH/0.1% (v/v) Tween-20] were incubated with 50-100 M fatty acids or 30-100 M hydroperoxides for 40 min on ice; in trapping experiments the hydroperoxides were only incubated for 1 min. The reactions (0.3-0.5 ml) were terminated with 10 ml water and the metabolites were immediately extracted on octadecyl silica (SepPak/C 18 ). The latter was washed with water and retained metabolites were eluted with ethyl acetate (4 ml). After being evaporated to dryness under N 2 , the residue was dissolved in ethanol (50-100 l), and 10 l were subjected to LC-MS/MS analysis.
Nitrogen powder of mycelia of Z. tritici was homogenized in 0.1 M KHPO 4 buffer (pH 7.3)/2 mM EDTA/0.04% (v/v) Tween-20. The supernatant, after centrifugation at 16,200 g (10 min, 4°C) was incubated with 100 M fatty acids for 30 min on ice. The products were extracted as above.

LC-MS analysis
RP-HPLC with MS/MS analysis was performed with a Surveyor MS pump (ThermoFisher) and an octadecyl silica column (5 m; 2 × 150 mm; Phenomenex), which was eluted at 0.3-0.4 ml/ min with methanol/water/acetic acid, 750/250/0.05. The effluent was subject to electrospray ionization 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 (5 amu for analysis of 2 H-labeled metabolites and hydroperoxides), the collision energy at 35 (arbitrary scale), and the tube lens at about 110 V. PGF 1 was infused for tuning. Samples were injected manually (Rheodyne 7510) or by an autosampler (Surveyor Autosampler Plus; ThermoFisher).
Triphenylphosphine (TPP) in hexane was used to reduce hydroperoxy fatty acids to hydroxy fatty acids. NaBH 4 or NaB 2 H 4 in enzymes, at least one separate subfamily (Fig. 1B). Second, the sequence information deduced from the DOX and CYP domains did not allow any unambiguous conclusions on their dual catalytic activities. Third, C. immitis and Z. tritici are important pathogens, and characterization of novel enzymes might provide important information for future biological studies (1,2). Work with mycelia of C. immitis has caused fatal infections in laboratory personnel (21), but we assessed the oxidation of fatty acids by mycelia of Z. tritici.
The structure of the -ketols formed from 18:2n-6 were confirmed by reduction of the ketone to a hydroxyl group, hydrogenation of the 12Z double bond, comparison with the mass spectra of the [ 13 C 18 ]-labeled -ketol (27), and with the mass spectra of the -ketol formed from 18:1n-9.

Bioinformatics
The ClustalW algorithm was used for sequence alignments (Lasergene, DNASTAR, Inc.). The MEGA6 software was used for construction of phylogenetic trees with 200 bootstrap tests of the resulting nodes (28).

MS/MS and MS 3 analysis of unsaturated -ketols formed by 8R-DOX-AOS
The MS/MS spectra of unsaturated -ketols, which are formed from 9-hydroperoxy fatty acids, show only weak informative signals, whereas their MS 3 spectra are more characteristic (27). We therefore investigated the MS/MS and MS 3 spectra of -ketols derived from 8-hydroperoxy fatty acids. from their MS 2 spectra (m/z 419→full scan), which were as reported previously (32). Steric analysis showed that both 12-HETE and 8-HETE mainly consisted of the S stereoisomers, as judged from reanalysis with added authentic 12S-HETE and 8R-HETE. The minor product, 10-HETE, eluted on CP-HPLC mainly as a single isomer (95%), but the stereo configuration was not further investigated.
EGP91582 aligns with 8R-DOX-LDS, and this protein is a strong candidate for the observed 5,8-LDS activities. The oxidation at C-13 of 18:2n-6 and 18:3n-3 was likely due to the only LOX of Z. tritici (GenBank identification number: EGP90986), which belongs to the family of fungal iron LOX (Fig. 6B). The 13S-LOX of Z. tritici thus has the same are consistent with suprafacial hydrogen abstraction and oxygen insertion, whereas 8R-DOX catalyzes antarafacial hydrogen abstraction and oxygenation as discussed above (33).

Catalytic properties of recombinant EGP87976 (9R-DOX-AOS) of Z. tritici
Recombinant EGP87976 oxidized 18:2n-6 and transformed 9R-HPODE to two polar products, as judged by RP-HPLC-MS analysis (peaks I and II in supplemental Fig.  S2A). The MS/MS and MS 3 spectra were as reported for the and -ketols, respectively, of 9-HPODE-derived allene oxides (27). The 18:2n-6 was also oxidized to 9-HPODE, and steric analysis by CP-HPLC (Reprosil Chiral-AM) and MS 2 analysis showed that 9-HPODE consisted of the 9S and 9R stereoisomers in a ratio of 1:3 (supplemental Fig.  S2B). This underestimates the relative formation of the 9R stereoisomer as 9R-HPODE was further transformed to and -ketols as major products.
The 8R-DOX-AOS efficiently transformed 9R-HPODE to an epoxy alcohol as a major product, but an -ketol catalytic activity as reported for the LOX of Fusarium oxysporum and Pleurotus ostreatus (8,34).
A summary of the oxidation of 18:2n-6 by mycelia and by 8S-and 9R-DOX-AOS is shown in Fig. 6C. The DOX-AOS activities could not be detected in mycelia and may only be expressed in response to environmental stimuli in analogy with other secondary metabolites.
Hydrolysis of allene oxides in an excess of methanol will form methoxy derivatives. We incubated 8R-DOX-AOS for 1 min with excess 8R-HPODE, added 30 vol of methanol and let the hydrolysis proceed for 1 h. To facilitate the analysis, we reduced the -ketols with NaBH 4 . Analysis showed the presence of the expected products, 8,9-DiHOME and 8-methoxy-9-hydroxy-12Z-octadecenoic acid, in a ratio of 9:1 (Fig. 7B) These spectra were consistent with the proposed structure. The trapping experiment indicates a short half time of the unconjugated allene oxide, 9R(10)-EODE, as about 90% was hydrolyzed by water after 1 min of incubation (Fig. 7B).
We next analyzed the transformation of [ 18 O 2 ]8R-HPODE by 8R-DOX-AOS to determine the incorporation of 18 O into the -ketol (8-hydroxy-9-oxo-12Z-octadecenoic acid). The ketone at C-9 of the -ketol can be exchanged with water and we therefore reduced the products formed after 1 min with NaBH 4 . LC-MS analysis of erythro and threo 8,9-DiHODE in the full scan mode showed incorporation of one molecule of 18 O (Fig. 7D) and the MS 2 spectrum (m/z 315→full scan) showed a signal at m/z 189, which demonstrated the 18 O-label at C-9 (Fig. 7E). as this fatty acid is a poor substrate of LOXs and fungal 9Rand 9S-DOX-AOSs (17,42).
The 8R-and 8S-DOX-AOS can be aligned with 51% amino acid identity, but the four amino acids in the 8-DOX domains from the catalytic Tyr to the proximal His heme ligand are not identical. The consensus sequence TyrArg-PheHis of all 9S-and 9R-DOX domains is conserved in 8R-DOX-AOS, but it is replaced in 8S-DOX-AOS with the consensus sequence TyrArgTrpHis of 8R-DOX-LDS, 10R-DOX-(CYP), and 10R-DOX-EAS. 8S-DOX-AOS is, nevertheless, the first described enzyme with a catalytic 8S-DOX domain.
The substrate recognition sites (SRSs) 4 (or I-helices) of the 8R-and 8S-AOS domains differ. The hexamer SRS 4 sequence ValAlaThrGlnAlaGln of 8R-AOS aligns except for one or two positions (in bold type) with the SRS 4 sequence ValAlaAsnGln(Ala/Gly)Gln of 5,8-LDS [see (18,43)]. The CYP domain of 8S-DOX-AOS is catalytically related to 9S-DOX-AOS, as it transformed 9S-HPODE to an -ketol, but this relation is not evident from SRS 4 or other sequence alignments of 8S-and 9S-AOS domains.

DISCUSSION
Our main goal was to investigate the catalytic properties of three putative DOX-CYP fusion enzymes of C. immitis (EAS28473) and Z. tritici (EGP83657 and EGP87976). We report that two of the recombinant enzymes form novel allene oxides and they are named 8R-and 8S-DOX-AOS, respectively, whereas the third enzyme belongs to the 9R-DOX-AOS subfamily. The sequential oxidation of 18:1n-9 by 8R-and 8S-DOX-AOS to allene oxides and hydrolysis to -ketols are outlined in Fig. 8. We detected 5,8-LDS and 13S-LOX activities of mycelia of Z. tritici (Fig. 6C), which could be attributed to expression of EGP91582 and EGP90986, respectively.
wheat (22,23). Plants transform 13S-HPOTrE sequentially to allene oxides and to jasmonates, which act as growth and defense hormones (37,38). The fungal AOS domains can only be aligned with plant AOSs (CYP74) and related P450 with a low degree of amino acid identity (18). Fungal AOSs and CYP74 have likely evolved independently. The parallel evolution of 8R-, 8S-, 9R-, and 9S-DOX-AOSs in fungi and CYP74 linked to LOX in plants suggest that the allene oxides may be of biological importance. This is well-established in the pathway to jasmonic acid with potent actions in plants (37,38). Lasiodiplodia theobromae and a few other fungal pathogens overproduce jasmonic acid to induce pathological plant growth (45). Fungal jasmonates may be formed by the biochemical pathway in plants, but details are lacking (8,46). The fungal repertoire of oxylipins likely participates in the struggle between the pathogen and its host, as well as in reproduction and development (1).
In summary, we have characterized fatty acid oxygenases of Z. tritici and report biosynthesis of two novel allene oxides formed by 8R-and 8S-DOX-AOS of C. immitis and Z. tritici. Their natural substrates are likely unsaturated C 18 fatty acids, and the biological function of these secondary metabolites should be evaluated in the context of related allene oxides formed by plants. Fig. 9. Illustration of the sequential biosynthesis of allene oxides from 18:2n-6 by 8S-DOX-EAS (EGP83657) and the suprafacial and antarafacial oxidation mechanisms of 8-and 9-DOX-AOS. A: Overview of the sequential biosynthesis of allene oxides by 8S-DOX-AOS and important amino acid residues for catalysis. B: The 8S-and 8R-DOX-AOSs abstract the proS hydrogen at C-8, whereas 9S-and 9R-DOX-AOSs abstract the proR hydrogen at C-11. The figure illustrates that these proS and proR hydrogens have the same absolute configuration relative to the 9Z,12Z-pentadiene structure. The red and blue arrows indicate the direction of oxygen insertion at C-8 and C-9, respectively, in relation to the pentadiene structure. The S and R assignments are due to the Cahn-Ingold-Prelog nomenclature rule.