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

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

and a carbon-centered radical intermediate. The latter is converted to an allene oxide and P450 compound II is recycled to ferric heme (Fig. 1C). Allene oxides are precursors of JA, which is an important signal molecule in plant defense and development and is present throughout the plant kingdom (18). The P450 domain of LDS transforms 8R-HPODE to diols by intramolecular oxygen insertion following heterolytic cleavage of the hydroperoxide O-O bond with formation of compound I (Por ϩ ⅐ Fe(IV)ϭO) (8,16,17,19). The latter abstracts hydrogen at either C-7 or C-5 and catalyzes oxygen rebound with retention of the configuration (Fig. 1D) (19 -21). In this way, LDS transforms 8R-HPODE to 7,8-or 5,8-DiHODE. The latter was identified along with 8R-HODE as a sporulation hormone of Aspergillus nidulans (22).
Oxylipins are formed by plants and plant pathogens in a complex network of interactions (9). Expression of plant LOX is often augmented in defense reactions to fungal infections, e.g. rice 13S-LOX by the rice blast fungus (23). Fungal plant pathogens can secrete LOX, e.g. the root rot fungus of wheat and the stem rot fungus of rice. These enzymes may oxidize plant cellular lipids to reactive oxygen species with detrimental cellular effects (24). JA is a prototype defense molecule of virtually all plants (18), but it can also be formed by fungi. Its biosynthesis by the tropical and devastating plant pathogen Lasiodiplodia theobromae was discovered more than 40 years ago (25,26). L. theobromae also oxidizes 18:2n-6 sequentially to 9R-HPODE and 9(10)-EODE by its 9R-DOX and AOS activities (27). Research progress in the area has been hampered because of environmental safety restrictions. These regulations do not apply to A. terreus, which oxidizes 18:2n-6 sequentially to 9R-HPODE and 9(10)-EODE in analogy with L. theobromae (28). Plant AOS belong to the CYP74 family and transform hydroperoxy fatty acids formed by 9S-and/or 13S-LOX, but the AOS and 9R-DOX activities of L. theobromae and A. terreus have not been characterized.
A. terreus is infamous for therapy-resistant infections in immunocompromised patients but also renowned for industrial production of lovastatin (29). The secondary metabolism of aspergilli has many industrial and toxicological implications and has attracted considerable attention (30). The genomes of at least eight aspergilli have been sequenced. A. terreus was sequenced in 2005 (for a review, see Ref. 31).
The genome of A. terreus contains five genes with homology to DOX-CYP fusion enzymes of other aspergilli (28). A phylogenetic tree of DOX-CYP fusion proteins is shown in Fig. 2A. Two proteins aligned with the 5,8-LDS and 10R-DOX clusters, indicating their putative catalytic activities. The remaining three proteins appear to be DOX-CYP orphans inasmuch as they do not cluster with characterized enzymes. There is also at least one gene with homology to the DOX domain (ATEG_03580; Fig. 2), but it lacks the P450 domain. In addition, sequenced aspergilli contain about 110 -155 P450 genes (32).
COX and the N-terminal DOX domain of DOX-CYP fusion proteins contain Tyr and His residues in a characteristic catalytic motif, Tyr-(Arg/His)-Trp-His (33). The conserved His residue in this sequence is the proximal heme ligand, and the Tyr residue likely forms the catalytically important radical (1,7,34). This sequence is conserved in two orphans but modified by replacement of the Trp residue by Phe or Met residues in two of the sequences (Fig. 2B). Whether these substitutions affect heme oxidation and formation of the Tyr radical is unknown. The C-terminal domains of the DOX-CYP orphans contain a series of conserved residues in the Cys pocket region, including the heme thiolate ligand, but not the typical insertion loop of CYP74 (Fig. 2B).
We hypothesized that the 9R-DOX and AOS activities could be present in an orphan DOX-CYP fusion protein in analogy with the 8R-DOX and hydroperoxide isomerase activities of LDS. In that case, A. terreus could also provide a model for studies of the tentative evolution of DOX-CYP fusion proteins into 9R-DOX and AOS.
The objective of the present study was to clone and express all five DOX-CYP homologues and one DOX homologue and to determine whether they could catalyze the 9R-DOX or AOS activities of A. terreus. This objective was met as described in the following experiments with identification of the first fungal AOS. This discovery enabled us to compare the active sites of AOS and the P450 domain of 5,8-LDS by site-directed mutagenesis. A, metal center of sLOX-1 in the oxidized form. The three nitrogen and oxygen ligands are indicated. The oxidized form catalyzes proton-coupled electron transfer from bisallylic positions of fatty acids. B, compound I of COX-1 and the catalytic tyrosyl radical. The latter catalyzes proton-coupled electron transfer from bisallylic positions of C 20 fatty acids and from allylic positions of C 18 fatty acids. LDS, 10R-DOX, and ␣-DOX use a closely related oxidation mechanism. C, allene oxide synthase. This class III P450 of the CYP74 family catalyzes homolytic cleavage of dioxygen bonds. This forms P450 compound II and a substrate radical, which is dehydrated to an allene oxide with formation of ferric heme. Prostacyclin synthase (CYP8A1) uses a similar reaction mechanism. D, LDS. This class III P450 catalyzes heterolytic cleavage of the dioxygen bond of 8R-HPODE and formation of compound I, which catalyzes hydrogen abstraction from C-7 or C-5 with formation of P450 compound II, a transient substrate radical, and "oxygen rebound" (modified from Ref. 21).
Assay of Non-competitive D-KIE of 9R-DOX-The supernatant of nitrogen powder of mycelia was prepared as above and incubated in triplicate with 0.1 mM 18:2n-6 or [11,11-2 H 2 ]18: 2n-6 for 5 or 20 min on ice. 13-Hydroxyoctadecatrienoic acid was added as an internal standard. The D-KIE was assayed by LC-MS/MS analysis of the carboxylate anions after reduction to alcohols (m/z 295-296 3 full scan) with monitoring of m/z 171. These experiments were also performed by comparison of [11, H 2 ]18:2n-6 with [ 13 C 18 ]18:2n-6 to correct for the small amounts of endogenous 18:2n-6.
RNA Extraction and cDNA Synthesis-Total RNA was isolated by LiCl extraction as described (37) and treated with RQ1 RNase-free DNase (Promega). First strand cDNA was synthesized using reverse transcriptase (Superscript III) with random hexamer or oligo(dT) 20 primers according to the manufacturer's instructions.
Real Time PCR Analysis-Gene expression was studied by real time PCR with an iCycler (Bio-Rad) as described (28). cDNA of strain A1156 and primer concentrations of each reaction (25 l) were 200 and 500 nM, respectively. ␤-Actin (ATEG_06973) served as the reference gene. Primers are listed in supplemental Table S1 and were designed using the Primer3 (ATEG_04755, 00985, 02036, and 03171) and PrimerQuest software programs (ATEG_03580, 06973, and 03992) allowing distinction of whether amplicons originated from cDNA or contaminating genomic DNA. All samples and negative controls were analyzed in triplicates. Amplification of one single product of the expected size was confirmed by melting point analysis and gel electrophoresis on a 2% agarose gel. Data analysis providing C T values was carried out with the iCycler software (Bio-Rad), and values for primer efficiencies were obtained from the LinRegPCR software (38).
Cloning of ATEGs-ATEG_04755 and 03171 were cloned from A. terreus strain IBT1948, whereas ATEG_03580, 02036, 00985, and 03992 originate from strain A1156 (NIH2624). All genes were cloned by RT-PCR as explained in detail in the supplemental Methods. In short, amplified cDNA segments were ligated in pJet1.2/blunt to build the expression constructs. The open reading frames were ligated into pIZ/V5-His in-frame with the V5 epitope and the His 6 tag. All constructs were confirmed by sequencing. All cloning primers are listed in supplemental Table S2.
Expression in E. coli-Subcloning of open reading frames from pIZ/V5-His to pET101D-TOPO vectors was performed by PCR technology following Invitrogen's instructions. All primers are listed in supplemental Table S3. The amplicons were ligated into pET101/D-TOPO and introduced into BL21 cells by heat shock transformation. Cells were grown to an A 600 of 0.6 -0.8 in low salt LB medium, and protein expression was induced by 0.1 mM isopropyl 1-thio-␤-D-galactopyranoside. Cultures were grown for 5 h at room temperature with moderate shaking (ϳ100 rpm). Cells were harvested by centrifugation and sonicated (Bioruptor Next Generation, 10 ϫ 30 s, 4°C) as described (33). At least three independent expressions of each protein were analyzed.
Western Blot Analysis-Recombinant enzymes were separated by SDS-PAGE on 8% polyacrylamide gels prior to blotting to nitrocellulose membranes. For Western blot analysis, a C-terminal anti-V5 antibody served as the primary antibody, and a horseradish peroxidase-conjugated anti-mouse IgG served as the secondary antibody (33). Both antibodies were usually diluted 1:12,500 (in 15% nonfat milk). Chemiluminescence was used for detection (ECL Advance kit, GE Healthcare).
Site-directed Mutagenesis-Site-directed mutagenesis of ATEG_02036 and 5,8-LDS ATEG_03992 was performed according to the QuikChange protocol (Stratagene) in pIZ/V5-His constructs (33). Amplicons were obtained from 10 ng of template by Pfu DNA polymerase (16 cycles) before digestion with DpnI (2 h, 37°C). Amplification of one distinct PCR product was confirmed by agarose gel electrophoresis before heat shock transformation (NEB5␣). Primers containing the designated replacements are listed in supplemental Table S4. All mutations were confirmed by sequencing before subcloning to pET101D-TOPO vectors as described above.
Enzyme Assays-Recombinant proteins expressed in insect cells or in E. coli were incubated with 100 M fatty acids (18: 1n-9, 18:2n-6, 18:3n-3, or 20:4n-6) in triplicate for 30 min on ice. The reaction (0.3-0.5 ml) was terminated with ethanol (2-4 volumes), an internal standard (13-hydroxyoctadecatrienoic acid) was added in some experiments, and proteins were removed by centrifugation. The metabolites were extracted on octadecyl silica (Sep-Pak C 18 ), evaporated to dryness, and diluted in ethanol (40 - ml/min. The effluent was subjected to electrospray ionization in a linear ion trap mass spectrometer (LTQ, ThermoFisher) with monitoring of carboxylate anions. The heated transfer capillary was set at 315°C, the ion isolation width was set at 1.5 atomic mass units, the collision energy was set at 35 (arbitrary scale), and the tube lens varied between 90 and 120 V. A trihydroxy fatty acid (prostaglandin F 1␣ ; 100 ng/min) was infused for tuning.
Bioinformatics-Proteins were aligned with the ClustalW algorithm (DNA Star software). Phylogenetic trees were constructed with MEGA 4 software with bootstrap tests of the resulting nodes (39). The distance within branches is based on the number of expected substitutions per amino acid position.
Miscellaneous Methods-Fatty acid composition of A. terreus was determined in nitrogen powder of mycelia after alkali treatment (0.5 M KOH in 90% methanol, 70°C, 1 h) and extractive isolation (CHCl 3 /methanol) according to Bligh and Dyer (40). The carboxylate anions were analyzed by direct injection. Fig. 2, sequence similarities placed ATEG_04755 together with other 10R-DOX. ATEG_03171 can be aligned with 60% identity to ATEG_ 04755 (ClustalW algorithm), but it is not a member of the 10R-DOX cluster (see Fig. 2). However, it belongs with ATEG_04755 in the CYP6001C subfamily (41). In ATEG_03171, the heme thiolate ligand is retained in contrast to ATEG_04755 and 10R-DOX of Aspergillus fumigatus and A. nidulans (37,42) (Fig. 2B).

Bioinformatics-As outlined in
ATEG_03992 aligns with 5,8-LDS and belongs to the CYP6001A subfamily (41). ATEG_00985 can be aligned with 44% amino acid identity to ATEG_03992, but the former is classified as an orphan of the CYP6002 family (41).
The ATEG_02036 sequence is relatively distant from the other DOX-CYP sequences and belongs to the CYP6003 family (41). The conserved DOX motif, Tyr-(Arg/His)-Trp-His, of COX, LDS, and 10R-DOX is modified by replacement of Trp by Phe in ATEG_02036 (Fig. 2B). This Trp residue is replaced by Met in the DOX homologue ATEG_03580.
9R-DOX and AOS activities of nitrogen powder preparations were detected in the cytosol (100,000 ϫ g supernatant) and in membrane fractions, respectively (28). Differential centrifugation showed that 9R-DOX-AOS activity was enriched in membrane precipitates (from 1,000 ϫ g to 17,000 ϫ g) in comparison with these supernatants and efficiently linked to AOS as estimated with [ 13 C 18 ]18:2n-6 as a substrate.
Gene Expression Analysis by Real Time PCR-Real time PCR revealed that all six studied genes were transcribed (Table 1). ATEG_06973, coding for ␤-actin, served as the reference gene. mRNA and cDNA were prepared from nitrogen powder of A. terreus with prominent 9R-DOX and AOS activities.
The values shown in Table 1 represent the median results of three analyses performed on different cDNA preparations and suggest that the genes were transcribed in the following order: ATEG_00985 ϭ 04755 Ͼ 02036 Ն 03992 Ն 03580 Ͼ 03171. The C T value for ATEG_03992 combines the transcripts of both the full length and its splice variant described below. For all primer pairs, the negative controls showed no significant amplification. We conclude that all six genes were transcribed and without large differences in mRNA expression levels (see Table 1).  ATEG_03992_sv (see below). These data were in agreement with the expected sizes of these proteins with the appended C-terminal His tags and V5 epitopes (ATEG_04755 (129 kDa), ATEG_03171 (126 kDa), ATEG_03992 (124 kDa), ATEG_03992_sv (122 kDa), ATEG_00985 (120 kDa), ATEG_02036 (133 kDa), and ATEG_03580 (74 kDa). We investigated the recombinant proteins for enzymatic activities. The results are summarized in Table 2. ATEG_02036 transformed 9R-HPODE to an allene oxide, but it did not oxidize 18:2n-6. We could identify 5,8-LDS, a splice variant of 5,8-LDS with 8R-DOX activity, 10R-DOX, and a homologue of 10R-DOX, ATEG_03171. The latter transformed 18:2n-6 to 10R-HPODE only in low yields (supplemental Fig. S3). 5,8-LDS and 10R-DOX are homologues to these enzymes of A. fumigatus and A. nidulans (17,20). ATEG_00985 formed only traces of 8R-HPODE (supplemental Fig. S3). Oxidation of C 18 fatty acids by ATEG_03580 could not be detected. We confirmed that the lack of activity was not due to the absence of heme by adding 1.5 M hematin back.

Summary of Cloning, Expression, and Western Blot Analysis-
AOS (ATEG_02036)-Cloning of ATEG_02036 yielded a 3432-bp cDNA fragment with 10 introns as predicted by conceptual translation (GenBank TM accession number EAU36998) and without nucleotide substitutions.
The ␣and ␥-ketols originate from spontaneous hydrolysis of an unstable allene oxide, 9(10)-EODE (28). This was con- ATEG_02036 and CYP6003B2 (41) are designated AOS for simplicity. We observed that C1073S replacement in AOS abolished the catalytic activity and confirmed expression of the mutant by Western blot analysis (supplemental Fig. S2).
Cloning and Expression of ATEG_00985 (Orphan), 03171 (10R-DOX), and 03580 (Orphan)-The results are summarized in Table 2, and details are provided in the supplemental Methods.
Site-directed Mutagenesis of the I-helices in the P450 Domains-AOS (ATEG_02036) and 5,8-LDS (ATEG_03992) can be aligned with ϳ38% amino acid identity (ClustalW) as shown in supplemental Fig. S6. Inspection of this alignment revealed a sequence of 12 amino acids that could be realigned as shown in Fig. 5 with 50% identity, including Asn 964 of AOS and Asn 878 of 5,8-LDS.
CYP74A and CYP8A1 catalyze homolytic cleavage of the O-O bonds. The marked Asn residues of these class III P450 in Fig. 5 are important for this process (8,47). These two Asn residues align with Thr in P450cam and prostaglandin H 19-hydroxylase (CYP4F8) using ClustalW. The Thr residue with an Asp residue forms the acid-alcohol pair, Asp-Thr, in the conserved sequence (Gly/Ala)-Gly-Xaa-(Asp/Glu)-Thr, which takes part in the heterolysis of O-O bonds by many class II P450 (12).
We replaced Asn 964 of AOS with Val and Asp, designated N964V⅐AOS and N964D⅐AOS, respectively. Both mutants had lost almost all AOS activity in comparison with native AOS (Fig. 6, A-C). Further analysis of N964D⅐AOS by NP-HPLC revealed that in addition to biosynthesis of the ␣-ketol there  terreus marked with asterisks (*) were mutated. Conserved residues are blue, and the Asn residues are red. The alignment of the last four proteins was obtained by ClustalW. The important Asn residues of the two class III P450, CYP74A and CYP8A1, are red, and other conserved residues are blue. The Thr residues of the two class II P450, P450cam and CYP4F8 (prostaglandin H 2 19-hydroxylase), align with the two Asn residues. The acid-alcohol pair, Asp-Thr, of P450cam and CYP4F8 in the conserved sequence Gly-Gly-Xaa-Asp-Thr are blue.
was also formation of epoxy alcohols ( Fig. 4 and supplemental Fig. S7). The homolytic cleavage of the hydroperoxide might be partly retained, but the desaturation step in biosynthesis of 9(10)-EODE appeared to be deranged with formation of epoxy alcohols (supplemental Fig. S7).
Three additional replacements, T957A⅐AOS, S959A⅐AOS, and Q967L⅐AOS, had little influence on the catalytic profile (see supplemental Fig. S7). This was unexpected inasmuch as the homologous Gln 881 residue of 5,8-LDS was important for the P450 activity and for the position of ferryl oxygen insertion (see below).
The partial sequence of the I-helix of 5,8-LDS in Fig. 5 contains several polar amino acids of possible catalytic importance, and we analyzed three of them by mutagenesis (Asn 878 , Gln 879 , and Gln 881 ). Q879L⅐5,8-LDS had little influence on the product profile, but replacements of Asn 878 and Gln 881 resulted in reduced catalytic activities and different product profiles.
N878L shifted oxygenation from C-5 to C-6 and to C-7 as shown by NP-HPLC (Fig. 7A). The efficient heterolytic cleavage of the O-O bond was not altered as only small amounts of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid were detected.
Replacement of Q881L⅐5,8-LDS almost abolished the diol synthase activity (Fig. 6D). The dramatic effect of Q881L was investigated by chain shortening of Gln to Asn and by replacement of the amide of Gln with a carboxyl group (Glu), with Asp, and with an amine (Lys) (Fig. 7).
We conclude that polar substitutions of Gln 881 yielded three main effects. First, the efficient heterolytic cleavage of 8R-HPODE can be partly replaced by homolytic cleavage as judged from formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid. Second, the position of oxygen insertion was partly shifted from C-5 to C-11, C-6, and C-7. Third, Q881E formed the erythro and threo isomers of the 8(9)-epoxy alcohol in a ratio of 4:1, indicating enzymatic biosynthesis.

DISCUSSION
We have cloned and expressed the first AOS of fungal origin, which constitutes our major finding. AOS of A. terreus was located in the P450 domain of a DOX-CYP fusion protein (ATEG_02036). One enzyme with 5,8-LDS activity and two with linoleate 10R-DOX activities were also expressed. Sitedirected mutagenesis of the I-helices of AOS and 5,8-LDS revealed catalytically important residues for homolytic and heterolytic scission of the O-O bond and for subsequent dehydration or hydroxylation (Figs. 8 and 9).
AOS-The amino acid sequence of AOS of A. terreus can be aligned with less than 25% amino acid identities to plant CYP74A and CYP8A1 but with ϳ38% identity to 5,8-LDS. It lacks the characteristic insert of nine amino acids in the con- served heme binding sequence of CYP74A (Fig. 2B) (8, 15). Site-directed mutagenesis revealed that the heme thiolate ligand (Cys 1073 ) was absolutely required for AOS catalysis.
The I-helices of AOS, 5,8-LDS, CYP74A, and CYP8A1 can be aligned with a conserved Asn residue (Fig. 5). The effects of site-directed mutagenesis of AOS and 5,8-LDS are summarized in Fig. 9.
Replacement of Asn 964 with Val or Asp residues in the Asn 964 -Val-Leu-Gln sequence markedly reduced the AOS activity. Asn residues of CYP8A1 and CYP74 are known to interact with the dioxygen of prostaglandin H 2 and hydroperoxides as judged by three-dimensional structure analysis and site-directed mutagenesis (8,47). Chain elongation of Asn 321 of CYP74A to Gln and replacement with Ala markedly reduced the AOS activity (8). Asn 321 of CYP74A has been suggested to form hydrogen bonds with the hydroperoxide and facilitate homolytic cleavage (8). This mechanism could be conserved in the fungal AOS. The activity of N964D⅐AOS was reduced and formed only traces of 9(10)-EODE (Figs. 6 and 9 and supple-   The two residues are present in the sequence Asn-(Val/Gln)-(Leu/Gly)-Gln (Fig. 5). The native catalytic activities of AOS and 5,8-LDS and the relative changes caused by the mutations are schematically graded from ϩϩϩ to Ϫ for simplicity. mental Fig. S7). Thus, the amide group of Asn 964 of the fungal AOS was not absolutely required for catalysis in analogy with Asn 321 of CYP74A.
AOS of the CYP74 family can metabolize 9-and/or 13-hydroperoxides of 18:2n-6 and 18:3n-3 (15). The transformation of 13S-hydroperoxyoctadecatrienoic acid to an allene oxide is the first step in the sequential biosynthesis of JA, including transformation by allene-oxide cyclase and ␤-oxidation (18). In contrast, the fungal biosynthesis of JA remains enigmatic.
The tropical plant pathogen L. theobromae and a few other fungi secrete JAs (25,26,48). A key intermediate in plant JA biosynthesis, 12-oxo-10,15(Z)-phytodienoic acid, was recently identified in the culture medium of L. theobromae (49). Plant and fungi also form JA with identical absolute configuration (25,48,50). The pathway to JA described in plants seems therefore likely, but key enzymes have not been detected in vitro (27). L. theobromae expresses 5,8-LDS and 9R-DOX linked to AOS in analogy with A. terreus (27). The genome of L. theobromae has not yet been sequenced; therefore, it is unknown whether it contains a homologue of AOS of A. terreus. Homologues with 64 -79% amino acid identities to ATEG_02036 are present in Aspergillus niger (see Fig. 2), Aspergillus flavus, Aspergillus oryzae, and Aspergillus kawachii, but the Asn 964 -Val-Leu-Gln sequence of AOS of A. terreus (Fig. 5) is modified in the position homologous to Asp-Val-Leu-(Gln/Asn) in these proteins. Their catalytic activities remain to be determined.

5,8-LDS-
The biological function of oxylipins produced by 5,8-LDS is well established in A. nidulans (7). The first step in biosynthesis of 5,8-DiHODE from 8R-HPODE is heterolytic cleavage of the dioxygen bond. In peroxidases, the distal histidine and an arginine residue are important for heterolysis of peroxides (51). P450cam and over 80% of human P450 contain the conserved sequence (Ala/Gly)-Gly-Xaa-Asp-Thr (12). The two latter residues are known as the "acid-alcohol" pair (12). This pair participates in transfer of hydrogen and in hydrogen bond networks, which facilitate heterolysis of the dioxygen bond (12).
Homolytic cleavage of 8-HPODE with formation of epoxy alcohols can be catalyzed non-enzymatically and to equal amounts of erythro and threo isomers (52). In contrast, P450 transform hydroperoxides after homolytic cleavage to epoxy alcohols with stereospecificity (52). N878L⅐5,8-LDS altered the substrate position for hydroxylation, and replacements of Gln 881 partly shifted the heterolytic scission of the O-O bond to homolytic as summarized in Fig. 9 with formation of erythro and threo isomers of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid in variable ratios. Gln 881 of 5,8-LDS in the Asn-Gln-Gly-Gln 881 sequence may have a function similar to that of the acid-alcohol pair Asp-Thr (12,51).
LOX with catalytic iron and DOX of the peroxidase family catalyze antarafacial hydrogen abstraction, and their D-KIEs for hydrogen abstraction at C-11 of 18:2n-6 differ by 1 order of magnitude (43)(44)(45). The D-KIE of 9R-DOX was low, consistent with oxidation by a tyrosyl radical and not by a LOX. Furthermore, the genome of A. terreus lacks LOX homologues. There-fore, we expected to find the 9R-DOX activity in one of the expressed DOX, but this was not the case. 9R-DOX catalyzes suprafacial hydrogen abstraction and oxygenation (28) and differs from 5,8-LDS, 10R-DOX, and COX-1 in this respect. 9R-DOX could nevertheless belong to the same DOX family as 8R-and 10R-DOX inasmuch as minor steric changes can alter their product profiles. 8R-DOX of A. fumigatus oxygenates the 9-trans-12-cis isomer of 18:2 by hydrogen abstraction at C-11 and oxygenation at C-9 to 9R-HPODE as the main metabolite (44), and replacement of one amino acid of 10R-DOX shifts oxygenation from C-10 to C-8 (37). In addition, the suprafacial hydrogen abstraction and oxygenation by 9S-MnLOX can be altered by a single amino acid replacement (53).
In conclusion, allene oxides of A. terreus are formed from exogenous 9R-HPODE by the P450 domain of a fusion protein of the DOX-CYP family. The reaction mechanism is closely related to plant AOS with homolytic cleavage of 9-HPODE and a catalytically important Asn residue in the heme environment. In analogy, a Gln residue of 5,8-LDS appears to facilitate heterolytic scission of 8R-HPODE and subsequent hydroxylation.