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

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Linoleate Diol Synthase Related Enzymes of the Human Pathogens Histoplasma Capsulatum and Blastomyces Dermatitidis Archives of Biochemistry and Biophysics 696 (2020) 108669 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi 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).
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