Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata)

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.


Introduction
Lancelets or amphioxi are the only living cephalochordates [1]. These organisms, as well as the urochordates (sea squirts) and vertebrates (including the jawless lamprey and hagfish), belong to the Chordata phylum [2][3][4][5]. Despite the separate evolution of cephalochordate and vertebrates from a common ancestor (which existed more than 520 million years ago), their morphology maximally retained characteristics of vertebrate ancestor Haikouella lanceolata [6,7]. Lancelets are considered to be intermediate between vertebrates and invertebrates. Thereby, these organisms are widely used as a model object to study the evolution of invertebrates and the origin of vertebrates [7].
Only a few Metazoan proteins of the CYP74 clan have been described until now. These are the ApAOS of the stony coral Acropora palmata [15], BfEAS (CYP440A1) of the lancelet Branchiostoma floridae [15], and NvEAS (CYP443D1) [23] and NvHPL/EAS (CYP443C1) [26] of the starlet sea anemone Nematostella vectensis. At the same time, the NCBI database contains numerous annotated but uncharacterized metazoan CYP74 clan members. Characterization of novel CYP74 clan member from lancelet species B. belcheri is reported in the present work. The protein exhibiting the mixed EAS/AOS activity is a first CYP74 clan enzyme of Chordata species possessing the AOS catalysis.

Bioinformatics' Analysis of the CYP440A18 Enzyme
The search for the putative genes of CYP74 clan in B. belcheri genome has been performed using TBLASTN and BLASTP tools at the NCBI database using BfEAS (CYP440A1; ACD88492.1) as query. The search revealed the target protein (458 aa; XP019641998.1) possessing 71% identity at 76% query coverage to BfEAS. Among green plants, the target protein possesses the highest identity to allene oxide synthases PpAOS2 (CYP74A8) of Physcomitrella patens (XP024372097.1) and LeAOS1 (CYP74A1) of Solanum lycopersicum (CAB88032.1): 26.95% and 26.86% identity at 83% and 75% sequence coverage, respectively. The name CYP440A18 has been assigned to this new sequence (Dr. David R. Nelson, personal communication).
Alignment of the CYP440A18 enzyme with other CYP74s revealed some features of primary structure typical for CYP74s such as the I-helix groove region (earlier "hydroperoxidebinding domain" [27], SRS-4), ERR-triad, PPV-domain, nine-amino acid insertion at the heme-binding domain, and cysteinyl heme ligand ( Figure 1).
The catalytically essential I-helix groove region has the V-M-F-N-A-V sequence. This region contains the conserved N residue (N223 in the investigated enzyme) involved in the initial stage of CYP74 catalysis, i.e., the homolytic cleavage of the O-O bond of fatty acid hydroperoxides [15]. Phylogenetic analyses using Clustal Omega and MEGA7 (the minimum evolution and the neighbor-joining methods) confirmed that the CYP440A18 enzyme belongs to the CYP74 clan. The epoxyalcohol synthase BfEAS (CYP440A1, GenBank: ACD42778.1) of lancelet B. floridae is the most similar to the CYP440A18 enzyme (bootstrap support is 100) (Figure 2). Thus, the structural and phylogenetic data confirmed that the CYP440A18 enzyme belongs to the CYP74 clan.
Alignment of open reading frame encoding the CYP440A18 enzyme with the genomic sequence mapped a gene of 5154 np length in a locus LOC109483426 (161,658-166,811, complement) and identified its structure. The coding sequence was interrupted into 5 exons by four introns. The target coding sequence adapted for expression in E. coli cells was custom synthesized by the Evrogen Company (Russia) and cloned into the vector pET-23a to yield the target recombinant protein with polyhistidine tag at C-terminus. His-tagged recombinant protein was obtained in BL21(DE3)pLysS strain cells (Novagen, USA) and purified by metal affinity chromatography. The enzymatic activity was controlled using the ultraviolet spectroscopy by the decrease of fatty acid hydroperoxide absorbance at 234 nm.

Analysis of Products of C 20 Hydroperoxide Conversions by the CYP440A18 Enzyme
Results of C20 hydroperoxide conversions by the CYP440A18 enzyme appeared similar to those of C18 hydroperoxide conversions. Thus, incubation of 15(S)-HPEPE ( Figure 6A) with the CYP440A18 enzyme resulted in formation of main product 9, the mass spectrum of which (Me/TMS, Figure 6B Catalytic hydrogenation of product 9 over PtO 2 followed by methylation and trimethylsilylation yielded saturated analogue, the mass spectrum of which corresponded to that of 13-hydroxy-14,15-epoxy-eicosanoic acid (Me/TMS). As a whole, the mass spectral data indicated the structure of 13-hydroxy-14,15-epoxy-5,8,11,17-eicosatetraenoic acid (Me/TMS) for compound 9. The minor product of this conversion was the α-ketol 10 ( Figure 6C

Formation of Cyclopentenones via Cyclization of Allene Oxides Biosynthesized from 13-HPOT and 15-HPEPE
It is noteworthy that the conversions of 13-HPOT and 15-HPEPE by CYP440A18 afforded not only α-ketols but also the cis-cyclopentenones 13 ( Figure 5A) and 14 ( Figure 6A Ratio of different reaction products of the CYP440A18 enzyme is summarized in Table 2. The products were quantified by integration of the total ion current GC-MS chromatograms.
Allene oxides (the primary AOS products) cyclization to cyclopentenones depends on the double bond in the β,γ-position towards the oxirane [36,37]. In other words, this β,γ double bond (the ω3 double bond in allene oxides formed from 13-HPOT and 15-HPEPE) shows the effect of neighboring group participation (anchimeric assistance) elevating the cyclization rate [36,37]. The recent DFT modelling revealed that the double bond-assisted oxirane opening is the rate-limiting step of the whole conversion of allene oxide to cyclopentenone [37]. Similarly, the higher outcome of the AOS (dehydrase) products upon the BbEAS/AOS incubations with 13-HPOT and 15-HPEPE (compared to 13-HPOD and 15-HPETE) suggests the impact of ω3 double bond on the conversion of the epoxyallylic radical intermediate. Presumably, the presence of ω3 double bond facilitates the elimination of hydrogen at the oxirane to form allene oxide. Similarly, the LuDES (CYP74B16) dehydrates the 13-HPOT predominantly to divinyl ether (ω5Z)-etherolenic acid, while 13-HPOD is largely isomerized to the hemiacetal (the HPL product) [29]. These observations indicate that the ω3 double bond affects the specificity of product formation by CYP74 clan enzymes.
The BbEAS/AOS is the second CYP74 clan member found in Chordata after the BfEAS (CYP440A1) of the lancelet Branchiostoma floridae [15]. At the same time, it is the first enzyme of Chordata possessing AOS activity. Overall, it is the second CYP74 clan enzyme in Metazoa possessing AOS activity, after the previously described allene oxide synthase ApAOS of stony coral A. palmata [15]. Besides the CYP74 clan proteins, there is a distinct kind of fatty acid hydroperoxide-metabolizing detected in soft corals and cyanobacteria. These are the catalase-related haemoproteins, for instance, the AOSs of soft corals Plexaura homomalla [38], Gersemia fruticosa [39], Capnella imbricata [40], and Acaryochloris marina [41] (see [42] for a review). All these enzymes are the fusion proteins consisting of the catalase and lipoxygenase domains. Thus, the detection of BbEAS/AOS adds more intricacy to the complex picture of oxylipin biosynthesis pathways in Chordata.

1.
The full-length coding sequence of Branchiostoma belcheri CYP440A18 enzyme has been expressed in Escherichia coli cells.

Bioinformatic Methods
The NCBI database was used for the search of the CYP74-related genes. Primer construction and multiple sequence alignments were performed using the Vector NTI program (Invitrogen, USA). The BLAST analyses of the CYP74s were performed using the protein BLAST tool. The multiple alignments of selected CYP74 amino acid sequences and phylogenetic tree building were made with MEGA7 software [45]. Multiple alignment was performed using the Muscle method, the phylogenetic tree was built by means of the maximum likelihood method based on the Poisson correction model [46], and the bootstrap consensus tree was inferred from 1000 replicates [47]. The analysis involved 54 amino acid sequences.

Expression and Purification of Recombinant Enzyme
The target sequence was adapted for obtaining recombinant enzyme in Escherichia coli cells and synthesized in ZAO Evrogen (Russia). The resulting sequence was cloned into the pET-23a (Novagen, USA) vector using NdeI and XhoI endonucleases to yield the target recombinant protein with His-tagged C-terminus. The resulting construction was transformed into Escherichia coli host strain BL21(DE3)pLysS (Novagen, USA). The heterologous expression of recombinant protein was performed in the LB/M9 mixed medium (1:1, by volume) supplemented with antibiotics at 37 • C until the cell culture reached an OD 600 of 0.6-0.8. The expression of the recombinant gene in E. coli cells was induced by adding 0.5 mM isopropyl-β-D-1-thiogalactopyranoside to the medium and the 5-aminolevulinic acid (100 mg/L), which facilitates the heme formation. The His-tagged recombinant protein was purified by immobilized metal affinity chromatography (IMAC) using Bio-Scale Mini Profinity IMAC cartridge and BioLogic LP chromatographic system (Bio-Rad, USA). The relative purity of the recombinant protein was measured by SDS-PAGE and staining of the gel with Coomassie brilliant blue R-250. Protein concentration was estimated as described before [48].

Kinetic Studies of Recombinant Enzyme
Enzymatic activity of the purified recombinant enzyme was determined by monitoring the decrease of the signal at 234 nm in a PB 2201 B spectrophotometer (ZAO SOLAR, Belarus) with substrate concentrations ranging from 5 to 150 µM. The analyses were performed in 0.6 mL of Na phosphate buffer (pH 6.0-8.0) at 25 • C. The initial linear regions of the kinetic curves were used to calculate the rates. The molar extinction coefficient for 9and 13-hydroperoxides of linoleic acid at 234 nm is 25,000 M −1 cm −1 . Kinetic parameters were calculated by fitting the datasets to a one-site saturation model for simple ligand binding using the SigmaPlot 11 software (Systat Software Inc., USA). Five independent experiments were performed for each specified variant.

Methods of Instrumental Analyses
The UV spectra of the reaction mixtures were scanned during the incubations of CYP440A18 with fatty acid hydroperoxides with Varian Cary 50 spectrophotometer. Alternatively, the UV spectra of products were recorded online during the HPLC separations using an SPD-M20A diode array detector (Shimadzu, Japan). Products (Me esters or Me/TMS derivatives) were analyzed by GC-MS as described previously [21]. GC-MS analyses were performed using a Shimadzu QP5050A mass spectrometer connected to a Shimadzu GC-17A gas chromatograph equipped with an MDN-5S (5% phenyl 95% methylpolysiloxane) fused capillary column (length, 30 m; ID 0.25 mm; film thickness, 0.25 µm). Helium at a flow rate of 30 cm/s was used as the carrier gas. Injections were made in the split mode using an initial column temperature of 120 • C, injector temperature 230 • C. The column temperature was raised at 10 • C/min until it reached 240 • C. Electron impact ionization (70 eV) was used.