Linoleate Diol Synthase Related Enzymes of the Human Pathogens Histoplasma Capsulatum and Blastomyces Dermatitidis

Archives of Biochemistry and Biophysics 696 (2020) 108669

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Linoleate diol synthase related enzymes of the human pathogens Histoplasma capsulatum and Blastomyces dermatitidis

Ernst H. Oliw

Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Box 591, SE 751 24, Uppsala, Sweden

ARTICLE INFO ABSTRACT

Keywords: Histoplasma capsulatum is an ascomyceteous fungus and a human lung pathogen, which is present in river valleys Cytochrome P450 of the Americas and other continents. H. capsulatum and two related human pathogens, Blasmomyces dermatitidis Linoleate diol synthase and Paracoccidioides brasiliensis, belongs to the Ajellomycetaceae family. The genomes of all three species code Lipid metabolism for three homologous and tentative enzymes of the linoleate diol synthase (LDS) family of fusion enzymes with Mass spectrometry dioxygenase (DOX) and cytochrome P450 domains. One group aligned closely with 8R-DOX-5,8-LDS of Asper Oxygenation mechanism Paracoccidioides brasiliensis gilli, which oxidizes linoleic acid to 5S,8R-dihydroxylinoleic acid; this group was not further investigated. The second group aligned with 10R-DOX-epoxy alcohol synthase (EAS) of plant pathogens. Expression of this enzyme from B. dermatitidis revealed only 10R-DOX activities, i.e., oxidation of linoleic acid to 10R-hydroperoxy-8E,12Z- octadecadienoic acid. The third group aligned in a separate entity. Expression of these enzymes of H. capsulatum and B. dermatitidis revealed no DOX activities, but both enzymes transformed 13S-hydroperoxy-9Z,11E-octade cadienoic acid efficiently to 12(13S)epoxy-11-hydroperoxy-9Z-octadecenoic acid. Other 13-hydroperoxides of linoleic and α-linolenic acids were transformed with less efficiency and the 9-hydroperoxides of linoleic acid were not transformed. In conclusion, a novel EAS has been found in H. capsulatum and B. dermititidis with 13S- hydroperoxy-9Z,11E-octadecadienoic acid as the likely physiological substrate.

1. Introduction oxidized fatty acids to the various end products of biological impor tance. For example, COX, 5-LOX, and leukotriene synthases in man Histoplasma capsulatum is an ascomyceteous fungus, which is found convert 20:4n-6 to pro-inflammatory prostaglandins and leukotrienes in river valleys worldwide [1]. Inhalation of spores of H. capsulatum can with pathophysiological roles in fever and asthma [8,9]. Furthermore, cause histoplasmosis, a lung disease [2]. Histoplasmosis may cause se 13S-LOX of some fungi oxidizes α-linolenic acid in the first step of vere symptoms in immunocompromized patients, but it is not a conta biosynthesis of jasmonates, which are powerful plant hormones with gious disease. Two closely related human pathogens are Blastomyces structural similarities to prostaglandins [10–14]. Fungi lack COX but can dermititidis and Paracoccidioides brasiliensis [3,4]. All three belong to the express related dioxygenases (DOX), which are often fused to cyto Ajellomycetaceae family and they can produce lung infections with chromes P450 (CYP) [7,15]. These fusion enzymes can oxidize unsatu spread to internal organs [5]. Interestingly, these fungi are thermally rated C18 fatty acids sequentially via hydroperoxides to diols, epoxy dimorphic, i.e., they can switch between growth as filamentousfungi at alcohols, and allene oxides [16–19]. Their N-terminal DOX domains ◦ ◦ 25 C and budding yeasts at 37 C [1–5]. form 8-, 9-, or 10-hydroperoxy metabolites by hydrogen abstraction, Oxylipins and eicosanoids designate oxygenated fatty acid metabo which is catalyzed by a Tyr radical in analogy with oxidation of 20:4n-6 lites derived from unsaturated C18 fatty acids and from arachidonic acid by COX, followed by insertion of molecular oxygen [20]. The C-terminal (20:4n-6), respectively [6,7]. Lipoxygenases (LOX) and heme-containing CYP domains with linoleate diol synthase (LDS), epoxy alcohol synthase dioxygenases (DOX) of the peroxidase-cyclooxygenase (COX) family (EAS), or allene oxide synthase (AOS) activities transform the hydro initiate oxygenation and downstream synthases then transform the peroxides of 18:2n-6 to various end products as illustrated in Fig. 1 [17,

Abbreviations: AOS, allene oxide synthase(s); COX, cyclooxygenase(s); CP, chiral phase; CYP, cytochrome P450; DOX, dioxygenase(s); EAS, epoxy alcohol syn thase(s); LDS, linoleate diol synthase(s); LOX, lipoxygenase(s); NP, normal phase; Ppo, psi producing oxygenase; Psi, precocious sexual inducer; RP, reversed phase; 9-H(P)ODE, 9-hydro(pero)xy-10E,12Z-octadecadienoic acid; 10-H(P)ODE, 10-hydro(pero)xy-8E,12Z-octadecadienoic acid; 10-H(P)OTrE, 10-hydro(pero)xy- 8E,12Z,15Z-octadecadienoic acid; 13-H(P)ODE, 13-hydro(pero)xy-9Z,11E-octadecatrienoic acid; 13-H(P)OTrE, 13-hydro(pero)xy-9Z,11E,15Z-octadecatrienoic acid. E-mail address: [email protected]. https://doi.org/10.1016/j.abb.2020.108669 Received 4 September 2020; Received in revised form 1 November 2020; Accepted 4 November 2020 Available online 13 November 2020 0003-9861/© 2020 The Author. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E.H. Oliw Archives of Biochemistry and Biophysics 696 (2020) 108669

Fig. 1. Overview of transformation of linoleic acid by fungal 9R- and 10R-DOX and by DOX-CYP fusion proteins: Abbreviations: AOS, allene oxide synthase; DOX, dioxygenase; LDS, linoleate diol synthase; EAS, epoxy alcohol synthase. Fig. 2. Phylogenetic tree of DOX-CYP fusion proteins of H. capsulatum, B. dermatitidis and P. brasiliensis with prototype enzymes with confirmed catalytic 18,21–25]. First identifiedwere 8R-DOX fused to linoleate 8R-DOX-5,8- activities as indicated. One sequence of B. dermatitidis, H. capsulatum, and and 8R-DOX-7,8-LDS. The former was found to produce precocious P. brasiliensis (marked green) aligns with 8R-DOX-5,8-LDS (PpoA), whereas sexual inducer (Psi) in Aspergillus nidulans and designated Psi producing alignment of the other six sequences of H. capsulatum, B. dermatitidis, and oxygenase A (PpoA) in recognition of the pioneering work of Champe P. brasiliensis suggested that they could belong two new subgroups (marked blue and coworkers [16,21]. These and homologous enzymes are now and red). The aligned sequences from GenBank are from top to bottom: grouped in the LDS family [26]. Some members may possess only one EGE79300, EEH08179, ODH49336, AGA95448, EDP50447, Q9UUS2, domain with catalytic activities. For example, the C-terminal domain of EHA52010, EGE82165, EEH06908, ODH51178, EHA53428, EJT82559, 9R-DOX shows insignificanthomology to CYP [27] and the CYP domain EAS28473, EGP83657, EHA25900, AGH14485, EGU88194, EFQ27323, of 10R-DOX (PpoC) of Aspergilli is inactivated due to a point mutation EEH05495, EGE81541, and ODH51007. The tree was constructed with Mn-LOX with replacement of the heme thiolate ligand [28]. Enzymes of the LDS of M. oryzae (AAK81882) as an outlier. (For interpretation of the references to family have been identifiedmainly from plant pathogens but also from a colour in this figure legend, the reader is referred to the Web version of this article.) few human pathogens (Aspergilli, Coccidioidis immitis) [7,25]. Our goal was to determine whether human pathogens of the Ajel lomycetaceae family might code for enzymes of the LDS family. Blast extraction kit was from Fermentas. RNaseA, lysozyme, and ampicillin analyses of the genomes of H. capsulatum, B. dermatitidis, and were from Sigma-Aldrich. The open reading frame of EEH05495 in P. brasiliensis revealed three groups of homologues. This is illustrated by pUC57 was ordered from GenScript (Piscatawy, NJ, USA) and trans a phylogenetic tree of these nine enzymes along with prototype pairs of ferred to the pET101/D expression vector (Champion pET Directional the LDS family (Fig. 2). One group, which is marked green in Fig. 2, TOPO Expression Kits, Invitrogen) using PCR primers from Eurofins appeared to align with little ambiguity to 8R-DOX-5,8-LDS (PpoA) of (Uppsala, Sweden). The reading frames of EGE82165 and EGE81541 in a Aspergilli. The second group (marked blue in Fig. 2) aligned with 10R- pET expression vector were ordered from VectorBuilder (Chicago, Ill, DOX-EAS of two plant pathogens. The third group (marked red) USA). appeared to form a separate branch. We report that the enzymes from H. capsulatum and B. dermatitidis of this group lacked DOX activities, but 2.2. Enzyme expression they transformed 13S-hydroperoxylinoleic efficiently to 12(13S)epoxy- 11-hydroxy-9Z-octadecenoic acid. The three pET plasmids were used to transform BL21 Star (DE3) E. coli (Invitrogen) by heat shock. Cells were grown until A600 of 0.6–0.8 2. Materials and methods from an overnight culture (5 ml Luria-Bertani broth with ampicillin), which was added to 2xYT medium (50–100 ml). 0.1–0.4 mM isopropyl- β 2.1. Materials -D1-thiogalactopyranoside was added to induce protein expression [19]. After 5 h under moderate shaking (~90 rpm) at room temperature, + ◦ 18:1n-9, 18:2n-6 (99%), and 18:3n-3 (99%) were from Larodan the cells were harvested by centrifugation ( 4 C; 20 min) and sus (Solna, Sweden) and Sigma-Aldrich (Darmstadt, Germany). 9R-, 9S-, pended in 50 mM Tris-HCl (pH 7.6)/5 mM EDTA/10% glycerol (v/v) – 13R-, and 13S-HPODE, and 13R- and 13S-HPOTrE were prepared by with lysozyme (1 2 mg/ml) on ice. The suspension was sonicated × + ◦ recombinant 9R-LOX (Anabaena [29]), 9S-LOX (tomato [30]), recom (Bioruptor Next Gen, 10 30 s, 4 C). Cell debris was removed by × + ◦ binant 13R-LOX (Magnaporthe oryzae [31]), and 13S-LOX (Lipidox, centrifugation (17,000 g, 20 min; 4 C). The supernatants were used ◦ Sigma). Epoxyalcohols were prepared from 13S-HPODE and 13S-HPO immediately or stored at 80 C. In some experiments, the supernatant TrE by treatment with hematin [32–34]. Cartridges with octadecyl silica was concentrated by diafiltration(Amicon, cut off at 50 kDa) with buffer change (0.1 M KHPO /2 mM EDTA/0.1% Tween-20 (pH 7.3). (Classic SepPak/C18) and silica (SepPak) were from Waters (Baltimore, 4 MD, USA). Phusion DNA polymerase and chemically competent Escher ichia coli (NEB5α) were from New England BioLabs (Ipswich, MA, USA). 2.3. Enzyme assay Restriction enzymes were from New England BioLabs and Fermentas (ThermoFisher Scientific, Uppsala, Sweden). Champion pET101D The supernatants from protein expression were incubated with 100 Directional TOPO Kit (Invitrogen) was from ThermoFisher. Gel μM fatty acids or with 20–100 μM hydroperoxy fatty acids for 30–40 min on ice. The reaction was terminated by addition of water (10 ml),

2 E.H. Oliw Archives of Biochemistry and Biophysics 696 (2020) 108669 followed by extractive isolation on SepPak/C18 cartridges [35]. The Table 1 latter were washed with water (2 ml) and eluted with 5 ml ethyl acetate. Alignment of important amino acid residues of four prototypes of the LDS family Water was removed and the organic phase was evaporated to dryness (in bold) with the nine tentative fusion enzymes of H. capsulatum. B. dermatitidis, and P. brasiliensis (with GenBank EEH, EGE, and ODH numbers, respectively). (N2); triphenylphosphine (3–5 μg) in hexane was added to reduce hy droperoxides to alcohols in some experiments. The samples were dis Enzyme Tyr and His ligand I-helix K-helix Cys pocket

– μ a solved in either 50 60 l ethanol for analysis by reverse phase (RP) 8R-DOX-5,8-LDS YRWH VANQAQ EGAR HQCLG chromatography or in 50–60 μl hexane/isopropanol, 9/1 (v/v), for EEH08179b YRWH VANQAQ EGSR HKCLG normal phase (NP) or chiral phase (CP) chromatography. EGE79300b YRWH VANQAQ EGTR HKCLG ODH49336b YRWH VANQAQ EGAR HKCLG 10R-DOXc YRWH VPAQAQ EAVR HGFLS 2.4. LC-MS analyses 10R-DOX-EASd YRWH VPNQAQ EGIR HACLG EEH06908e YRWH VPSQAQ EGLR FTCFG RP-HPLC with MS/MS analysis was performed with a Surveyor MS EGE82165e YRWH VPSQAQ EGLR FTCFG μ ODH51178e YRWH VPSQAQ EGIR FTCFG pump (ThermoFisher Scientific)and an octadecyl silica column (5 m; 2 f × 150 mm; Phenomenex (Torrance, CA, USA), which was usually eluted 9S-DOX-AOS YRFH GVPVTA EAQR HECIA EEH05495g YRFH GAAIST EMQR HQCFG at 0.3 ml/min with methanol/water/acetic acid, 750/250/0. The EGE81541g YRFH GAAIST EMQR HQCFG effluent was subject to electrospray ionization in a linear ion trap mass ODH51007g YRFH GAAIST EMQR HQCFG spectrometer (LTQ, ThermoFisher). The heated transfer capillary was set a ◦ 8R-DOX-5,8-LDS (PpoA) of Aspergillus fumigatus (EDP50447). at 310 C, the ion isolation width at 1.5 amu (5 amu for hydroperoxides), b Sequence homologues of 8R-DOX-5,8-LDS (PpoA); a translation error in the the collision energy at 35 (arbitrary scale), spray current and the tube 8R-DOX domain of EEH08179 was corrected (see Results). c lens at 4.5 kV and 72 V, respectively. PGF1α was infused for tuning. 10R-DOX (PpoC) of A. fumigatus (EDP52540); the CYP domain is inactivated Samples were injected manually (Rheodyne 7510) or injected by an auto by a point mutation with loss of the heme thiolate ligand (Cys residue). sampler (Surveyor Autosampler Plus, ThermoFisher). d 10R-DOX-EAS of M. oryzae (EHA53428). NP-HPLC and CP-HPLC with MS/MS analysis were performed as e Sequence homologues of 10R-DOX-EAS. f follows. The NP-HPLC column (5 μm; 2 × 250 mm; Kromasil 100 Si, 9S-DOX-AOS (EGU88194) of F. oxysporum. g Dalco Chromtech, Marsta,¨ Sweden) was eluted with hexane/isopropyl Sequence homologous of 9-DOX-AOS. alcohol/acetic acid, 97/3/0.01 at 0.5 ml/min for separation of erythro and threo trans-epoxy alcohols derived from 13-HPODE [33,36]. Isomers PheThrCys, which differs from the consensus sequence HisXxxCys of the of 13- and 10-HPODE were resolved by CP-HPLC on Reprosil Chiral-NR CYP domains of the LDS family (cf. Table 1). This replacement of His (5 μm; 2 × 250 mm; Dr. Maisch, Ammerbuch, Germany), eluted (0.3 residue with Phe might inactivate catalysis as did the replacement of His ml/min) with hexane/isopropyl alcohol/acetic acid, 98.5/1.5/0.02 with Ala of 8R-DOX-5,8-LDS (PpoA) [38]. (v/v/v) [37]. The effluentsof the NP- and CP-HPLC columns were mixed on-line with 0.15–0.2 ml/min isopropyl alcohol/water, 3/2 (v/v), and 3.1.3. Homologues with DOX-AOS and sterol C22 desaturase subject to electrospray ionization as described above. The three homologues enzymes of H. capsulatum, B. dermatitidis, and P. brasiliensis (EEH05495, EGE81541, and ODH51007, respectively) 2.5. Miscellaneous aligned in a distinct branch of the phylogenetic tree with 9R- and 9S- DOX-AOS as close neighbors (Fig. 2). Comparison with 9-DOX-AOS Sequences were aligned with the ClustalW algorithm and the showed that they also contained the YRFH sequence but they lacked phylogenetic tree was constructed with the Megalign software (DNAS in analogy with them characteristic amide residues in I helices of LDS TAR, Inc., USA). and EAS (Table 1). In addition, Blast analysis revealed sequence similarity with sterol 3. Results C22 desaturases. EEH05495 of H. capsulatum and EEQ92396 of B. dermatitidis could be aligned over the heme thiolate ligand of the 3.1. Amino acid sequence comparison sterol C22 desaturases (EEH05495 and EGE81541, respectively) with E values < 4e 5, but the alignments only covered 5% of their sequences. 3.1.1. Homologues of 8R-DOX-5,8-LDS ODH51007 of P. brasiliensis aligned with its sterol C22 desaturase 4 Amino acid sequence alignments showed that three putative DOX- (EEH16357) with an E value of 5e . CYP enzymes of H. capsulatum (EGE79300), B. dermatitidis Alignment of EEH05495 of H. capsulatum with EGE81541 of (EEH08179), and P. brasiliensis (ODH49336) aligned with two pro B. dermatitidis revealed two unique inserts in the DOX domain of totypes of 8R-DOX-5,8-LDS (Fig. 2). However, the published sequence of EEH05495, viz., nine residues between Ala470 and Gly480 and five B. dermatitidis (EEH08179) was likely incorrect due to a translation residues between Ser506 and Thr512. These inserts were not present in error. The corrected translation led to an insert of 31 amino acid residues DOX domains of prototypes of the LDS family. The inserts might alter between Asp202 and Leu208. This added sequence, SLLVRKRFRPH- catalysis and we therefore expressed both EEH05495 and EGE81541. PNKIISSMLFYLAASIIIHD, contains the distal heme ligand near its end, i.e., the His residue in the conserved sequence IIHD of the LDS family. 3.2. Catalytic properties of recombinant EGE82165 Furthermore, alignment of catalytically important amino acid residues of the three tentative enzymes with 8R-DOX-5,8-LDS strengthened the Recombinant EGE82165 (B. dermatitidis) oxidized 18:2n-6 to 10-H homology (Table 1). (P)ODE as the main product (Fig. 3A). Small amounts of 8-H(P)ODE were also detected, which is consistent with hydrogen abstraction at 3.1.2. Homologues of 10R-DOX-EAS C-8 by the Tyr radical and insertion of O2 at C-10 or at C-8. Three putative DOX-CYP enzymes of H. capsulatum (EEH06908), 10R-HPODE elutes after and 10S-HPODE elutes before 13S-HPODE B. dermatitidis (EGE82165), and P. brasiliensis (ODH51178) aligned in a on the Reprosil Chiral-NR HPLC column [37]. 10-HPODE formed by separate branch of the phylogenetic tree close to two prototypes of 10R- recombinant EGE82165 eluted after 13S-HPODE, and it was therefore DOX-EAS (Fig. 2). Alignment of important amino acid residues identified as 10R-HPODE (Fig. 3B). The MS/MS spectrum of 10-HODE confirmed the similarity of their DOX domains with both 8R-DOX-LDS showed characteristic signals at m/z 183 and 155 (Fig. 3C), whereas and 10R-DOX-EAS (Table 1), but a remarkable difference was present in the MS3 spectrum of 10-HPODE yielded characteristic signals at m/z their CYP domains. All three enzymes contained the sequence 211, 199, 153, and 139 (Fig. 3D). Significant formation of epoxy

3 E.H. Oliw Archives of Biochemistry and Biophysics 696 (2020) 108669

Fig. 4. RP-HPLC-MS3 analysis of the oxidation of 18:3n-3 at C-10 by recom binant EGE82165 (B. dermatitidis). A. LC-MS3 analysis (m/z 309 → 291→full scan): top trace, total ion current (TIC); bottom trace, selective ion monitoring of m/z 273 (291-18). B. MS3 spectrum of 10-HPOTrE (m/z 309 → 291→full scan). Epoxyalcohols were detected in peak I and 10-HPOTrE in peak II.

the amount of 10-HPOTrE in peak II (Fig. 4A, bottom trace). The MS3 spectrum of 10-HPOTrE differed from that of 10-HPODE with signals, inter alia, at m/z 209 and 197 (Fig. 4B), but the MS/MS spectrum of 10- HOTrE showed a similar fragmentation as 10-HODE. The 12Z double bond of 18:2n-6 and 18:3n-3 appeared to be essential as 18:1n-9 was not oxidized.

3.3. Catalytic properties of recombinant EEH05495 and EGE81541 Fig. 3. RP-HPLC-MS analysis of oxidation of 18:2n-6 at C-10 by recombinant EGE82165 (B. dermatitidis). A. LC-MS/MS and MS3 analysis of hydroperoxides 3.3.1. Recombinant EEH05495 (H. capsulatum) and alcohols revealed formation of 10-HPODE and 10-HODE. B. CP-HPLC-MS/ Recombinant EEH05495 did not possess detectable DOX activities MS analysis of 10-HPODE and 13S-HPODE revealed that the former eluted after with 18:2n-6 and 18:3n-3 as substrates, but it possessed prominent EAS 13S-HPODE; it was identified as the 10R stereoisomer as the 10S and 10R activities. Recombinant EEH05495 transformed 100 μM 13S-HPODE stereoisomer elutes before and after 13S-HPODE, respectively. C. MS/MS efficientlyto 12(13S)epoxy-11-hydroxy-9Z,11E-octadecadienoic acid as 3 spectrum of 10-HODE formed by EGE82165. D. MS spectrum of 10-HPODE the main products as judged from RP-HPLC-MS/MS analysis (Fig. 5A). formed by EGE82165. The MS/MS spectrum was as previously reported [33]. Steric analysis of the epoxy alcohol by NP-HPLC suggested that it consisted mainly of the alcohols derived from 10R-HPODE could not be detected, which sug less polar erythro stereo isomer (erythro/threo, 9/1) (Fig. 5B), as erythro gested low or absent EAS activities. stereo isomers of trans-epoxy alcohols are less polar than the corre 18:3n-3 was oxidized by recombinant EGE82165 in analogy with sponding threo isomers [36], but this assignment is uncertain if the 3 18:2n-6 to 10-H(P)OTrE (Fig. 4A). MS analysis (m/z 309 → 291→full epoxide is cis. 13R-HPODE was transformed in analogy with scan) with monitoring of m/z 273 suggested that formation of epoxy 13S-HPODE, but less efficiently (data not shown); 9S- and 9R-HPODE, alcohols, which eluted in peak I, were insignificantin comparison with 8R-HPODE, and 15S-HPETE were not transformed significantly.

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The transformations of 100 μM 13S-and 13R-HPOTrE are illustrated in Fig. 6A and B (after reduction of HPOTrE to HOTrE with triphenyl phosphine). The MS/MS spectrum of the main product, 12(13S)epoxy- 11-hydroxy-9Z,15Z-octadecadienoic acid was as previously reported (Fig. 6C) [33]; the MS3 spectrum yielded only intense signals in the upper mass range (Fig. 6D). 13S-HPOTrE appeared to be transformed more efficiently than 13R-HPOTrE as judged from the total ion in tensities of substrate end products in these parallel experiments, but this was not further investigated. 12(13S)Epoxy-11-hydroxy-9Z,15Z-octadecadienoic acids formed from 13S-HPOTrE were resolved into two stereoisomers on NP-HPLC in

Fig. 6. LC-MS/MS analysis of the transformation of 13S- and 13R-HPOTrE to 12(13S)epoxy-11-hydroxy-9Z,15Z-octadecenoid acids by the EAS activities of recombinant EEH05495 (H. capsulatum). The hydroperoxides were reduced to alcohols with triphenylphosphine before analysis. A. RP-HPLC-MS analysis and separation of products formed from 13S-HPOTrE. B. RP-HPLC-MS analysis of Fig. 5. Transformation of 13S-HPODE by recombinant EEH05495 products formed from 13R-HPOTrE. C. Partial MS/MS spectrum of 12(13S) (H. capsulatum). A. RP-HPLC-MS/MS analysis of HODE (top trace) and epox epoxy-11-hydroxy-9Z,15Z-octadecenoid acid. The inset illustrates formation of yalcohols (bottom trace) after reduction of hydroperoxides by triphenylphos the anions at m/z 197 and 227. D. Partial MS3 spectrum of 12(13S)epoxy-11- phine. B. Separation and analysis of two stereoisomers of 12(13S)epoxy-11- hydroxy-9Z,15Z-octadecenoid acid. The signal at m/z 263 (291-28) is likely due hydroxy-9Z-octadecenoic acid by NP-HPLC-MS/MS analysis; the erythro ste to loss of CO. E. NP-HPLC-MS/MS analysis of two stereoisomers of 12(13S) reoisomer of trans epoxy alcohols is less polar than the threo isomer. The ste epoxy-11-hydroxy-9Z,15Z-octadecadienoic acids formed from 13S-HPOTrE. reoisomers were labeled accordingly, but this assignment is uncertain if the Assuming a trans epoxide, the isomers were labeled erythro and threo based on epoxide is cis. their polarity, but this assignment is uncertain if the epoxide is cis. F. RP-HPLC- MS/MS analysis showed that the EAS transformation of 13S-HPOTrE to epoxy alcohols was retained after diafiltration (50 kDa).

5 E.H. Oliw Archives of Biochemistry and Biophysics 696 (2020) 108669 a ratio of 6:4 (Fig. 6E). Finally, recombinant EEH05495 with 1138 groups by sequence alignment (Fig. 2). We report that two of these en amino acids (128 kDa) was retained after diafiltration (50 kDa) with zymes, 5,8-LDS and 10R-DOX, have counterparts in Aspergilli and plant buffer change (Fig. 6F). pathogens, whereas the third possesses unique EAS activities. These findings are summarized in Fig. 8 and they are based on the following 3.3.2. Recombinant EGE81541 (B. dermatitidis) considerations. Recombinant EGE81541 did not oxidize 18:2n-6 and 18:3n-3 One group of enzymes (Fig. 2) was closely related to 8R-DOX-5,8- significantly, but 100 μM 13S-HPODE was transformed to 12(13S) LDS (PpoA) as judged by sequence homology. The distal and proximal epoxy-11-hydroxy-9Z-octadecenoic acid efficientlyas shown in Fig. 7A. His heme ligands and the catalytic Tyr residue of the DOX domains of the 13S-HPODE appeared to be the preferred substrate in comparison with LDS family were likely conserved along with known critical residues in 100 μM 13R-HPODE, which yielded both 12 (13R)epoxy-11-hydroxy- the CYP domain (Table 1). Catalytic activities cannot be unambiguously 9Z-octadecenoic and 9-hydroxy-12 (13R)epoxy-10E-octadecenoic acids predicted by sequence homology. It nevertheless seemed unlikely that (Fig. 7B); the latter eluted first and it was identified by MS/MS as recombinant expression of these homologues could be justified as they described [33]. 13S-HPOTrE was transformed to 12(13S)epoxy-11-hy appeared to be very close to well characterized enzymes (Fig. 2; droxy-9Z,15Z-octadecadienoic acid (Fig. 7C), but in contrast to Table 1). EEGH05495, the transformation of 100 μM 13R-HPOTrE was insignifi The second group showed sequence homology with 10R-DOX-EAS of cant and without stereospecificity( Fig. 7D). 9S-and 9R-HPODE were not M. oryzae and G. graminis (Fig. 2), but recombinant expression revealed transformed. We conclude that the 13S-HPODE is the preferred only 10R-DOX activities. This discrepancy might be related to the substrate. PheThrCys sequence of the CYP domain, which contains the heme thi olate ligand. This corresponds to the strictly conserved HisXxxCys 4. Discussion sequence of members the LDS family with catalytically active CYP do mains. Substitution of the conserved His residue by Phe is thus un Our aim was to characterize the enzymes of the LDS family of three precedented, but Brodhun et al. found that replacement with Ala closely related human fungal pathogens: H. capsulatum, B. dermatitidis, abolished the 5,8-LDS activity of 8R-DOX-5,8-LDS (PpoA) of A. nidulans and P. brasiliensis. These tentative enzymes fell into three separate [38]. Replacement of His with Phe might therefore explain the lack of EAS activities. In analogy, the CYP domain of 10R-DOX of A. fumigatus is inactivated by replacement of the heme thiolate ligand with Phe (Table 1). It will be interesting to determine whether these substitutions, Phe to His and Phe to Cys, respectively, can restore catalytic activities of these CYP domains. The third group of Fig. 2 provided the most unexpected findings.The expressed enzymes of H. capsulatum and B. dermatitidis did not oxidize linoleic and α-linolenic acids to 13S-hydroperoxides. This could be due to lack of incorporation of heme in their DOX domains or other expression errors, but their CYP domains at the other end of the

Fig. 7. LC-MS/MS analysis of the transformation of 13S- and 13R-HPODE and 13S- and 13R-HPOTrE by recombinant EGE81451 (B. dermatitidis). The samples were analyzed after reduction of the hydroperoxides to alcohols. A. RP-HPLC- MS/MS analysis of the transformation of 13S-HPODE by MS/MS analysis of Fig. 8. Overview of the proposed oxidation of linoleic acid and the trans 13-HODE (top trace), epoxy alcohols (middle trace), and by selective ion formation of 13S-HPODE by B. dermatitidis, H. capsulatum, and P. brasiliensis. A. monitoring of m/z 197 of 12 (13)epoxy-11-hydroxy-9Z-octadecenoic acid Oxidation of linoleic acid by the tentative 8R-DOX-5,8-LDS was deduced by (bottom trace). B. RP-HPLC-MS/MS analysis of the transformation of 13R- sequence homology, and oxidation by 10R-DOX was deduced by the recombi HPODE as in A. C. RP-HPLC-MS/MS analysis of the transformation of 13S- nant enzyme of B. dermatitidis. B. Transformation of 13S-HPODE by the re HPOTrE with monitoring of 13-HOTrE (top trace), epoxy alcohols (middle combinant EAS of B. dermatitidis and H. capsulatum. The cis and/or trans trace), and selective monitoring of m/z 197 of 12 (13)epoxy-11-hydroxy- configurationof the 12(13S)epoxide has not been determined. Assuming a trans 9Z,15Z-octadecadienoic acid (bottom trace). D. RP-HPLC-MS analysis of the epoxide, NP-HPLC analysis suggests that the relative C11 and C12 configuration transformation of 13R-HPOTrE with top, middle, and bottom traces as in C. is erythro, but this is uncertain if the epoxide is cis.

6 E.H. Oliw Archives of Biochemistry and Biophysics 696 (2020) 108669 expressed proteins possessed EAS activities. The latter transformed 13S- [2] G.S. Deepe Jr., Outbreaks of histoplasmosis: the spores set sail, PLoS Pathog. 14 hydroperoxides to 12(13S)epoxy-11-hydroxy metabolites; this is the (2018), e1007213, https://doi.org/10.1371/journal.ppat.1007213. http://www. ncbi.nlm.nih.gov/pubmed/30212569. firstreport of these EAS activities of the LDS family. It will be of interest [3] J.A. Smith, G. Gauthier, New developments in blastomycosis, Semin. Respir. Crit. to determine whether this enzyme is expressed in mycelia with a func Care Med. 36 (2015) 715–728, https://doi.org/10.1055/s-0035-1562898. http tional DOX domain. ://www.ncbi.nlm.nih.gov/pubmed/26398538. [4] R. Martinez, New trends in paracoccidioidomycosis epidemiology, J. 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