Catalytic and Structural Properties of Heme-containing Fatty Acid Dioxygenases: Similarities of Fungal Dioxygenases and Cyclooxygenases

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(209) List of Papers. This thesis is based on the following publications, which are referred to in the text by their Roman numerals:. I. Garscha, U., Oliw, E.H. (2008) Pichia expression and mutagenesis of 7,8-linoleate diol synthase change the dioxygenase and hydroperoxide isomerase. Biochemical and Biophysical Research Communications, 373, 579-83. II. Garscha, U., Oliw, E.H. (2008) Critical amino acids for the 8(R)dioxygenase activity of linoleate diol synthase. A comparison with cyclooxygenases. FEBS Letters, 582, 3547-51. III. Garscha, U., Jernerén, F., Chung, D., Keller, N.P., Hamberg, M., Oliw, E.H. (2007) Identification of dioxygenases required for Aspergillus development. Journal of Biological Chemistry, 282, 34707-18. IV. Garscha, U., Oliw, E.H., (2007) Steric analysis of 8-hydroxy- and 10hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases. Analytical Biochemistry, 367, 238-46. V. Garscha, U., Oliw, E.H. (2009) Leucine/valine residues direct oxygenation of linoleic acid by (10R)- and (8R)-dioxygenases. Expression and site-directed mutagenesis of (10R)-dioxygenase with epoxyalcohol synthase activity. Journal of Biological Chemistry, 284, 13755-65. Reprints were made with permission from the copyright holders..

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(211) Contents. Introduction...................................................................................................11 Oxylipins ..................................................................................................11 Heme-containing dioxygenases................................................................14 Prostaglandin H synthases ...................................................................15 Linoleate diol synthases.......................................................................19 Alpha dioxygenases .............................................................................21 Cytochrome P450 and hydroperoxide isomerase .....................................22 Aims..............................................................................................................24 Methods ........................................................................................................25 Expression systems ..................................................................................25 Expression in Pichia pastoris....................................................................25 Expression vector.................................................................................26 Transformation and expression of 8-DOX in P. pastoris ....................26 Expression in insect cells .........................................................................26 The baculovirus based system .............................................................26 The plasmid based expression system .................................................28 Enzyme activity assay ..............................................................................29 Site-directed mutagenesis.........................................................................29 Western blot analysis ...............................................................................29 Synthesis of racemic hydroxy- and dihydroxy fatty acids .........................30 High performance liquid chromatography ...............................................30 Mass spectrometry....................................................................................31 Results...........................................................................................................32 Recombinant 7,8-LDS..............................................................................32 Site-directed mutagenesis of 8-DOX (papers I and II) ............................32 Dioxygenases in Aspergilli (paper III) ....................................................34 Catalytic mechanism (paper III)..........................................................35 Identification of dioxygenases (paper III) ...........................................35 Stereochemistry of mono- and dihydroxy metabolites (paper IV) ...........36 Cloning and expression of 10-DOX (paper V) ........................................37 Catalytically important residues for dioxygenation at C-8 and C-10 (paperV) ...................................................................................................38.

(212) Discussion .....................................................................................................40 Conclusions...................................................................................................47 Acknowledgements.......................................................................................48 References.....................................................................................................50.

(213) Abbreviations. AA AOX COX CP-HPLC DiHODE DOX HETE HODE HPL HPLC HPODE KODE LA LDS LOX LT MBD NP-HPLC NSAID ODA PG PGH PGHS PGI2 PIOX ppo psi RP-HPLC RT-PCR TXA2 YPDS. arachidonic acid alcohol oxidase cyclooxygenase chiral phase-high performance liquid chromatography dihydroxyoctadecadienoic acid dioxygenase hydroxyeicosatetraenoic acid hydroxyoctadecadienoic acid hydroperoxide lyase high performance liquid chromatography hydroperoxyoctadecadienoic acid ketooctadecadienoic acid linoleic acid linoleate diol synthase lipoxygenase leukotriene membrane binding domain normal phase-high performance liquid chromatography non-steroidal anti-inflammatory drug oxydecenoic acid prostaglandin prostaglandin H prostaglandin H synthase prostacyclin pathogen-inducible oxygenase psi producing oxygenase precocious sexual inducer reverse phase-high performance liquid chromatography reverse transcription polymerase chain reaction thromboxane A2 yeast extract-peptone-dextrose-sorbitol medium.

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(215) Introduction. Oxylipins Oxylipins are mono- and dioxygenated polyunsaturated fatty acids, which often act as lipophilic signaling molecules. These oxylipins are formed enzymatically by mono- and dioxygenases, but also non-enzymatically by autoxidation [1]. Oxygenated fatty acids are widely distributed in plants, vertebrates, and fungi [2-7]. Mammalian oxylipins Oxylipins formed in mammals include the vast group of the eicosanoids (Fig. 1), which are lipid mediators deriving from C20 fatty acids. The most important is arachidonic acid (AA). Arachidonic acid is esterified in membrane phospholipids, and released by cytosolic phospholipase A2 after diverse extracellular stimuli (e.g., mechanical trauma, inflammation, cytokines) and MAP kinase activation [8]. Subsequently, AA is transformed by different enzymes, including dioxygenation by prostaglandin H synthase (PGHS) and lipoxygenases (LOX) [2, 9]. Monooxygenation occurs by cytochrome P450 leading to hydroxy- and epoxy fatty acids (Fig. 1). Prostanoids with ring structures (prostaglandin, prostacyclin), and thromboxanes are derived from the key intermediate prostaglandin H2 (PGH2), which is formed from AA by PGHS. AA can be transformed by the lipoxygenase pathway to hydroperoxides (HPETE) and leukotrienes (leukocyte-derived-trienes) [10-12]. In general, lipoxygenases (LOX) recognize a 1,4-pentadiene structure of polyunsaturated fatty acids, and form hydroperoxides, containing a conjugated double bond system [13]. AA is converted to 5S-HPETE, and further to an epoxide leukotriene (LTA4) by 5-lipoxygenase (5-LOX). A leukotriene hydrolase metabolizes LTA4 to LTB4, which is a potent leukocyte chemo attractant, and involved in a number of inflammatory processes. LTA4 can also be conjugated with glutathione to LTC4, which is further metabolized to LTD4 and LTE4 [14, 15]. These leukotrienes, were previously known as “slow-reacting substances of anaphylaxis” (SRS-A) [16]. They are synthesized by mast cells, eosinophils, and macrophages, and act as bronchoconstrictors and proinflammatory mediators [17].. 11.

(216) Figure 1. Overview of eicosanoids formed in man.. In addition to 5-LOX, 12- and 15-LOX are identified in man, and oxidize AA at C-12 and C-15, respectively [18]. According to the phylogenetic analysis, human lipoxygenases can be divided into three subgroups, (i) the leukocyte-type 12S-LOX and reticulocyte-type 15S-LOX, (ii) epidermal 12R/15S-LOX, and (iii) the platelet-type 12S-LOX [18]. Although the catalytic mechanisms of LOXs are well studied, less is known about the biological function. As described above, leukotrienes, formed by 5-LOX, are related to anaphylactic and inflammatory diseases, especially to asthma. Their action can be blocked by 5-LOX inhibitors (e.g. Zileuton), or by antagonism of the leukotriene receptor (Montelukast, Zafirlukast) [19]. 12/15-LOX metabolites presumably have effect on atherogenesis [20, 21], and 12R-LOX and epidermal LOX (eLOX3) are involved in formation of the water-impermeable barrier of skin, by production of hepoxilins (epoxy-hydroxy fatty acids), and mutations conclude in congenital ichthyosis [22-24]. In addition, 15-LOX is involved in mitochondria degradation, which is essential for erythropoiesis. Hydrophilic pores are formed by oligomerization and integration of the enzyme in the membrane [25, 26]. Therefore, 15-LOX is associated with natural membrane degradation processes, e.g., during reticulocyte maturation.. 12.

(217) Plant oxylipins Oxylipins in plants emerge from oxidation of polyunsaturated fatty acids. Different from mammalian oxylipins, most often C18 fatty acids function as source material. Plants lack a protective immune system, but it is well established that oxylipins take part in a complex defense system as signal molecules and protective compounds. Formation of oxygenated fatty acids can occur enzymatically or non-enzymatically. Enzymatically generated phyto-oxylipins can be obtained by three different pathways: (i) biosynthesis by lipoxygenases (LOX), (ii) formation by cytochrome-P450 dependent monooxygenases, and (iii) by heme-containing fatty acid dioxygenases [5, 27-29]. Plant LOXs dioxygenate C18 fatty acids with a 1,4-pentadiene structure to a hydroperoxide after double bond migration [28]. According to the position of dioxygenation of LA (linoleic acid) and -LA (-linolenic acid), plant LOXs are classified as 9-LOX and 13-LOX [28]. The most intensively characterized enzyme is the soybean lipoxygenase-1 (sLOX-1), which forms 13S-HPODE, and is used as a model enzyme of this group [30, 31]. The hydroperoxides are further metabolized by diverse enzymes, including allene oxide synthase (AOS), hydroperoxide lyase (HPL), or epoxy alcohol synthase (EAS). These enzymes belong to the CYP74 family [5, 32]. Plant cytochrome P450 enzymes convert saturated and unsaturated fatty acids to hydroxy- and epoxy metabolites. Less is known about the biological function; however, it is believed that the enzymes prevent the plant from accumulation of free fatty acids [5]. Dioxygenation at the C-2 of fatty acids is catalyzed by alphadioxygenases (-DOX) with homology to PGHS [27]. They belong to the heme-containing dioxygenases, and are thoroughly described below. Non-enzymatically formed oxylipins are racemic, and are generated by autoxidation during oxidative stress and formation of reactive oxygen species (ROS) [1]. Phytoprostanes or C18-isoprostanes are prostaglandin-like compounds, which are formed non-enzymatically and are induced by stress [33, 34]. Autoxidation products are found in healthy plant tissues, and are increased in older plants as shown in Arabidopsis thaliana [35]. Fungal oxylipins The present study focuses on oxylipins formed by fungal dioxygenases, in comparison to PGHS. Therefore special attention is given to the following section. The fatty acid profile can differ between distinct phyla, but ascomycetes and basidomycetes contain predominantly C18 fatty acids [36]. Thus, most of the oxylipins derive from linoleic and -linolenic acids. Mono- and dihydroxy metabolites of LA and -LA were identified in many fungi. 8R-hydroxyoctadecadienoic acid (8R-HODE) was first detected 13.

(218) in the basidomycete Laetisaria arvalis, and given the trivial name laetisaric acid [37]. A few years later, Champe et al. identified psi factors (precocious sexual inducers) of A. nidulans as 8R-HODE and 5S,8R-DiHODE, but the mechanism of biosynthesis remained unknown [38, 39]. During the following years, 8R-HODE was also discovered in many other fungi, for example, Gaeumannomyces graminis [40], Magnaporthe grisea [41], Agaricus bisporus [42], and in distinct Aspergilli species [43]. In addition to 5S,8RDiHODE formed by A. nidulans, other dihydroxy metabolites were found in fungi. G. graminis and M. grisea produced 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) [41, 44]. 8,11-DiHODE was generated by A. bisporus [42] and the sewage fungus Leptomitus lacteus [45]. Lipoxygenases are also present in some fungi. 9- and 13-hydroxyoctadecadienoic acids (9-HODE, 13-HODE), were isolated from the ascomycete Fusarium oxysporum [46, 47]. The formation of 11S- and 13R-HODE by G. graminis is very well studied, since they are produced by a unique manganese lipoxygenase [48, 49]. As an uncommon property, the enzyme contains manganese in the catalytic center, instead of iron. Similar to other LOX, the formation of oxylipins starts with a hydrogen abstraction at a bisallylic carbon. However, oxygen insertion occurs in a suprafacial way, which differs from other LOXs [50]. Candida albicans and Cryptococcus neoformans, as well as a number of other pathogenic fungi, have been reported to transform exogenous AA to prostaglandins [51, 52]. The enzymes forming these eicosanoids have not been identified. It is suggested that the pathogenic fungi converted host derived AA. Furthermore, the prostaglandins were detected by ELISA [51], and cross reactivity to non-enzymatically formed isoprostanes might be possible. On the other hand, mass spectrometry identified PGE2 produced by C. neoformans [53]. Due to the lack of enzymes and the precursor compound, the formation of prostaglandins is still under discussion. So far, all fungal oxylipins are formed by heme, and non-heme dioxygenases. During the 90s, 7,8-linoleate diol synthase (7,8-LDS) was discovered, which forms 8R-HODE and 7S,8S-DiHODE. This enzyme was characterized, and belongs to the group of heme-containing dioxygenases, and is thoroughly described below.. Heme-containing dioxygenases Sequence alignment of 7,8-LDS of G. graminis revealed homology to PGHS. This was also found to be the case with oxygenases of Aspergilli. All enzymes belong to the group of heme-containing dioxygenases of the myeloperoxidase family [54]. The next chapter will focus on the dioxygenase PGHS, as it is the best studied enzyme and a pharmaceutical target.. 14.

(219) Prostaglandin H synthases Historical Background Prostaglandins were first isolated from sheep vesicular glands in the 1930s by von Euler [55], but it took 30 years to determine the structure and the mechanism of biosynthesis of prostaglandins [56]. The development and improvement of mass spectrometry facilitated the breakthrough, and until now, this method is of invincible importance for analyzing oxygenated fatty acids. During the 70s prostaglandin H synthase was purified, and two distinct activities were discovered, the cyclooxygenase and the peroxidase activity [57-60]. In 1971, aspirin and non-steroidal anti-inflammatory drugs (NSAID) were shown to inhibit the prostaglandin biosynthesis [61]. Consequently, the analgesic, anti-inflammatory, and antipyretic action of this drug could be explained as well as many side effects. These findings stimulated research in the field of prostaglandins. In 1982, Sune Bergström, Bengt Samuelsson and Sir John Vane received the “Nobel Prize in Physiology and Medicine” for their discoveries concerning prostaglandins, leukotrienes, and related biologically active substances, and the mechanism of action of NSAIDs. Until the early 90s, there was belief in the existence of only one PGHS. However, independent groups identified a second isozyme of PGHS, which is inducible expressed [62-65]. During the following years, PGHS-1 and PGHS-2 were crystallized with different ligands, and the catalytic structure could be extensively studied [66-70]. Mechanism of enzyme catalysis Prostaglandin H synthases are bifunctional enzymes, and convert arachidonic acid sequentially to prostaglandin G2 (PGG2) by cyclooxygenase activity, and further to prostaglandin H2 (PGH2) by peroxidase activity [71, 72] (Fig. 2). PGHS is commonly known as cyclooxygenase (COX), however, the name displays only one enzyme activity. The enzyme reaction starts by a two electron oxidation of the ferric heme to compound I, an oxyferryl group plus a cationic protoporphyrin radical. This compound is reduced by a single electron traveling from Tyr385, resulting in a tyrosyl radical and an intermediate II (oxyferryl group plus a neutral protoporphyrin) [73-75]. The tyrosyl radical initiates the conversion of AA by stereospecific abstraction of the pro-S hydrogen at C-13 [76]. Molecular oxygen is inserted in an antarafacial way at C-11, and an 11R-hydroperoxyl radical is formed. Subsequently, this radical attacks C-9, in order to form an endoperoxide and a radical at C-15. A second dioxygenation occurs, and PGG2 is obtained. PGG2 is reduced by the peroxidase activity to PGH2 [71].. 15.

(220) Figure 2. Biosynthetic pathway of PGHS.. Structure and catalytically important residues Crystallization of PGHS, complexed with different substrates, gave insights about the tertiary structure and about stereochemical aspects [66-68, 77, 78]. PGHSs are homodimers, located on the luminal side of the endoplasmatic reticulum, and the dimerization is necessary for enzyme catalysis [79]. Each monomer consists of three domains, an epidermal growth factor (EGF) at the N-terminus, a membrane binding domain (MBD), and a large C-terminal catalytic domain [70]. The EGF forms the substantial part of the dimer interface, but the functional importance remains unclear. The MBD contains 4 amphipathic -helices, and their hydrophobic and aromatic residues protrude from the helices, and form a hydrophobic surface. This allows interaction with the membrane lipid bilayer [66]. The catalytic domain of PGHS consists of -helices and shares homology with other mammalian myeloperoxidases [54]. COX and peroxidase (POX) are located apart from each other, but are catalytically connected, as POX activity is necessary to activate COX. The peroxidase is located on the other site of the MBD and is defined by a heme prosthetic group. Additional, hydrophobic residues create a “dome” over the heme, but they do not appear to be important for the POX activity 16.

(221) [80]. The ligand proximal to the heme is His388, which has been subjected to site-directed mutagenesis. His388Gln and His388Ala lost COX and POX activities [81-83]. His207 was found to be at the distal site of heme and is of fundamental importance for hydroperoxide reduction [84]. The entrance to the COX site is located between the MBD helices. The cyclooxygenase active site forms a long, hydrophobic channel. Crystal structures and a series of mutational studies give detailed information about the catalysis and interaction of amino acid residues with the substrate [70, 85]. It could be demonstrated that Arg120 of PGHS-1 binds the carboxylate of AA and the carboxylate of certain PGHS-1 inhibitors (e.g. flurbiprofen) [86-88]. In contrast, mutation of homologous arginine of PGHS-2 had little effect on COX and POX, which suggests other residues involved in substrate binding [89]. When AA enters the active site of COX, C-13 is located close to the phenolic oxygen of Tyr385. As described above, it is evident that the tyrosyl radical 385 stereospecifically abstracts the pro-S hydrogen at C-13, and thus initiates the catalytic reaction [90, 91]. Tyr504 was suggested to be an alternative tyrosyl radical in PGHS-2 [92]. Tyr504Phe had little effect on COX and POX activity. Therefore Tyr504 is apparently not essential for enzyme catalysis. Curiously, Tyr504Phe of PGHS-1 lacked, but Tyr504Ala retained COX and POX activity [81]. Additionally, C-11 of AA is surrounded by Val349, Ala527, Ser530, and Leu531. They form a small pocket in which oxygen can migrate [86]. Val349 appeared important for stereospecific dioxygenation of AA in the active site [85, 93]. PGHSs convert AA to metabolites with 11R- and 15Sconfiguration with one exception. PGHS from Plexaura homomalla forms 15R-prostaglandins [94]. This PGHS conserves an isoleucine in homologous position of Val349. Mutational studies confirmed the importance of this residue, as Val349Ile formed 35% 15R-prostaglandins, and reverse mutation of Ile349Val of PGHS of P. homomalla generated 70% of 15S-PGs [95]. The Val349Leu mutant formed a 65:35 mixture of 15S/R-HETE [85]. Furthermore, replacement by smaller hydrophobic residues (Ala, Ser, Thr) increased formation of 11R-HETE in comparison to PGG2 [85]. Ser530 is the well known residue, which is irreversible acetylated by aspirin [77, 96, 97]. This results in total loss of PGHS-1 activity. Interestingly, acetylation of Ser530 in PGHS-2 abolished PG synthesis, but retained 15HETE formation [97]. The active site in PGHS-2 is larger than in PGHS-1, and that might explain why PGHS-2 can accommodate an acetyl group [67, 68]. Moreover, Ser530 controls the stereochemistry at C-15. Acetylated PGHS-2 forms 15-HETE with R configuration [97]. 15R-PGs were obtained, when Ser530 was replaced by Met, Thr or Val in PGHS-2 [93]. Corresponding mutations in PGHS-1 resulted in inactive enzymes. Additional to Val349 and Ser530, Tyr348 seems to be important for positioning of AA at the active site. Crystal structures of PGHS-1 suggested that the hydroxy group of Tyr348 forms a hydrogen bond to Tyr385, and brings 17.

(222) Tyr385 in right position for hydrogen abstraction. Furthermore, the aromatic ring of Tyr348 is in van der Waals distance to C-12, C-13, and C-14 of AA, which proposed hydrophobic interactions. Tyr348Phe, with associated loss of the hydroxyl and putative hydrogen bond to Tyr385 had little effect on cyclooxygenase activity, and formation of a tyrosyl radical similar to the native enzyme was shown [98]. However, replacement by leucine resulted in loss of cyclooxygenase activity, and suggested movement of C-13 of AA away from Tyr385 [85]. Within the Y385HWH motif, Trp387 makes van der Waals interaction with Tyr385 [86] and is located close to C-11 of the bound AA. Therefore, this residue is important for proper cyclization and stabilization of the endoperoxide, and further generation of PGs. Trp387Phe reduced formation of PGs, but increased formation of 11-HETE [85, 99]. Two isoforms of PGHS As mentioned above, in the early 90s, a second isoform was discovered, and showed ~65% sequence identity with PGHS-1 [71]. In general, PGHS-1 is a constitutively expressed enzyme, whereas PGHS-2 is inducible after inflammation and other stimuli [100]. PGHS-2 is also constitutively expressed in kidney and brain [101, 102]. The cyclooxygenase active site of PGHS-2 is larger and of slightly different shape [67]. These differences are due to three amino acid distinctions between PGHS-1 and -2 (Ile523Val, Ile434Val, His513Arg), and enable synthesis of specific PGHS-2 inhibitors, also known as coxibs. Differences also occur in the membrane binding domains [103]. PGHS-2 contains a larger opening and Arg120, important for substrate binding in PGHS-1, is displaced [67]. Furthermore, the C-terminus of PGHS-2 contains an 18 residues long cassette, which is probably responsible for rapid proteolysis. An additional N-glycosylation at Asn594 allows the enzyme to enter the endoplasmatic reticulum-associated degradation system [104, 105]. Inhibition of PGHS PGHS-1 and -2 can be inhibited by NSAIDs (non-steroidal antiinflammatory drug). All NSAIDs compete with AA for the cyclooxygenase binding site of PGHS. Ibuprofen, naproxen, indometacin and flurbiprofen are example for reversible binding inhibitors. Due to inhibition of TXA2 biosynthesis in platelets and decreased formation of protective PGs, classical NSAIDs cause serious adverse effects, predominantly gastrointestinal bleeding and ulcer. Selective PGHS-2 inhibitors (celecoxib, valdecoxib, lumiracoxib, and etoricoxib) have anti-inflammatory, analgesic, and anti-pyretic activities without gastrointestinal complications. Their selectivity towards PGHS-2 is based on structural differences in the COX site. The more bulky residue in PGHS-1 in position 523 (Ile) does not allow access of larger inhibitors [106, 107]. Other strategies, like neutraliza-. 18.

(223) tion of carboxyl goups of classical NSAIDs resulted in selective inhibition of PGHS-2 [108]. In 2004, long-term treatment with coxibs was associated with an increased cardiovascular risk. Therefore rofecoxib was withdrawn from the market. A possible explanation could be the inhibition of prostacyclin (PGI2) formation by PGHS-2 in the endothelium, but unaffected thromboxane A2 (TXA2) production by PGHS-1 in platelets [109, 110]. Prostaglandin synthases Prostaglandin H2, formed by PGHS is further metabolized by different enzymes resulting in formation of PGs, PGI2, and TXA2. While the latter are formed by P450 enzymes, prostaglandins are synthesized by certain enzymes, including PGE-, PGD-, and PGF synthases. Already 1976, ChristHazelhof could demonstrate conversion of PGH2 to prostaglandin E2, F2 and D2 by glutathione-S-transferase in the presence of glutathione [111]. PGE2, the most common prostaglandin in man, is formed by three different PGE synthases, the cytosolic PGE synthase (cPGES), and two membrane-bound PGE synthases (mPGES-1 and -2). They belong to the MAPEG family (membrane-associated protein involved in eicosanoid and glutathione metabolism) [112, 113], and require glutathione as an essential cofactor. Interestingly, cPGES and mPGES2 are constitutively expressed, and isomerizes predominantly PGH2 formed by PGHS-1, whereas mPGES-1 converts PGH2 produced by the inducible PGHS-2 [114].. Linoleate diol synthases In the early 90s, Brodowsky et al. showed that G. graminis transforms 18:2n-6 to 8R-hyrdoperoxyoctadecadienoic acid (8R-HPODE) [40], and they could demonstrate that the hydroperoxide is further metabolized to 7S,8Sdihydroxyoctadecadienoic acid (7S,8S-DiHODE) [115]. Finally, Su purified and characterized this enzyme, which is known as 7,8-linoleate diol synthase (LDS) [116]. 7,8-LDS converts linoleic acid to 8R-HPODE by dioxygenase activity, and further to 7S,8S-DiHODE by isomerase activity [44] (Fig. 3). The catalytic mechanism starts with oxidation of the resting ferric heme to compound I (oxyferryl heme + protoporphyrin radical cation), which is able to oxidize a neighboring tyrosine to a radical. This tyrosyl radical could be confirmed by electron paramagnetic resonance spectroscopy (EPR) [117], and abstracts the pro-S hydrogen at C-8 from linoleic acid. Molecular oxygen is inserted in an antarafacial way, and lead to formation of 8R-HPODE. The hydroperoxide is either reduced to the alcohol 8R-HODE, or transformed to 7S,8S-DiHODE via pro-S hydrogen abstraction at C-7 and suprafacial hydroxylation.. 19.

(224) Figure 3. Reaction mechanism of 7,8-LDS.. When 7,8-LDS was cloned, sequence alignment revealed homology with PGHS [118]. In 2005, the genomes of A. nidulans and A. fumigatus were published [119, 120], and three homologous gene sequences to 7,8-LDS were identified in each strain. Already 20 years ago, Champe et al. detected oxylipins as precocious sexual inducers (psi) [38, 39]. The homologous gene sequences of the putative oxygenases were named ppoA, ppoB, and ppoC (psi producing oxygenase) [121]. However, how these oxylipins were formed remained unknown. Biological importance of oxylipins formed by LDS The understanding of the biological function of fungal oxylipins is now emerging. It is believed that these compounds have impact on the development, on the communication with the host on a cellular basis, and on reproduction. When 8R-HODE was identified, Bowers and coworkers could show that this oxygenated fatty acid could inhibit the growth of the oomycete Pythium ultimum, in comparison to the untransformed fatty acid [37]. At the same time, 8-HODE and 5,8-DiHODE were identified as precocious sexual inducers (psi), which inhibited asexual and induced sexual sporulation in A. nidulans [39]. When the genome of Aspergilli was published, Keller and coworkers used the 7,8-LDS sequence to identify related oxygenases, proposed to produce these psi factors [121]. Three putative oxygenases were detected in the genome of Aspergilli and in order to elucidate the biological importance of oxylipins, ppoA, ppoB and ppoC were deleted in Aspergilli [122, 123]. Overexpression of PpoA, resulted in an increased level of 8-HODE and elevated sexual spore development, whereas deletion of ppoA resulted in. 20.

(225) decrease of 8-HODE and increased asexual sporulation [122]. Deletion of ppoC resulted in alteration of the phenotype, as the conidia grow faster in shaking and slower in stationary cultures in comparison to the wild type [124]. Furthermore, the ppoC-strain showed increased oxidative stress tolerance and elevated susceptibility to phagocytosis [124]. The three Ppo enzymes are not expressed constitutively. For example, PpoB is expressed at undetectable levels in certain strains, except under special growth conditions, whereas PpoC is expressed at high levels, and for longer time periods than PpoA [121, 125]. This suggests a complex network of regulation. Oxylipins probably regulate the formation of secondary metabolites. Mutant strains ppoAppoc and ppoAppoBppoC were unable to produce the mycotoxine sterigmatocystein, and resulted in overproduction of penicillin [126]. Therefore, manipulation of Aspergilli may increase the production of pharmaceuticals. LOX produced oxylipins (9-H(P)ODE and 13-H(P)ODE) are known to be communication molecules. Exogenous 13S-HPODE inhibits sexual spore production in A. nidulans [127], and Aspergilli infection induced peanut LOX (PnLOX) in seeds, which forms 9- and 13-HPODE [128]. Recently, the reciprocal crosstalk between Aspergilli and seeds was shown to be governed by fungal oxylipins [129]. Aspergilli with defective oxylipin formation (ppo) could not induce LOX expression in peanut seeds, and A. nidulans, which hosts a plant LOX could increase the conidia production [129].. Alpha dioxygenases Heme-containing dioxygenases were discovered in diverse plants. They belong to a group of enzymes, which dioxygenate fatty acids at C-2, and are therefore called -dioxygenases (-DOX) [6]. The first -DOX was a pathogen induced oxygenase (PIOX), which was discovered in tobacco [130]. This PIOX showed sequence homology to PGHS [131]. During the following years, -DOX was identified in e.g., Arabidopsis [130], cucumber [130], rice [132], pea [133], and Ulva pertusa [134]. These -DOXs abstract the pro-R hydrogen from C-2 of C16 and C18 fatty acids and insert molecular oxygen with retention of the configuration, resulting in formation of an unstable hydroperoxide [6]. In aqueous solution, the hydroperoxide decays spontaneously to an aldehyde. -DOX1 of Arabidopsis and of rice are structurally characterized, and heme ligands and putative tyrosine residues for radical formation are identified [132, 135]. The moos Physcomitrella patens dioxygenate predominantly C16 fatty acids to the 2-hydroxy fatty acids, but C18 fatty acids were poor substrates. This suggests an -DOX, which is distinct from -DOX of tobacco [6]. In Arabidopsis, a second -DOX sequence was found with homology to the -DOX of P. patens. This group is defined as -DOX2.. 21.

(226) -DOX1 is induced by pathogen attack [136, 137]. In contrast, microbial infection did not induce the gene of -DOX2 in Arabidopsis. However, gene transcripts were increased after leaf detachment [6].. Cytochrome P450 and hydroperoxide isomerase As mentioned in the first chapter, AA can be transformed to hydroxy- and epoxyeicosatetraenoic acids (HETE, EET) by heme-containing monooxygenases [138-141]. This includes (i) -hydroxylation, which is predominantly achieved by the CYP4 family [138], (ii) epoxidation generated by CYP2 enzymes [142], and (iii) allylic hydroxylation with or without double bond migration [138, 141]. All of these CYP450 reactions need the presence of NADPH, functioning as a reductant. However, already in the early 80s Ullrich et al. discovered cytochrome P450 enzymes, which transform peroxides [143, 144]. These enzymes metabolize the endoperoxide PGH2 by rearrangement of the oxygen to thromboxane by CYP5A, and to prostacyclin by CYP8A. During the 90s, cytochrome P450 enzymes were discovered in plants. They metabolize peroxides, which are formed by LOX. These uncommon cytochrome P450 enzymes do not need NADPH as a reductant, which is essential for other CYP 450s [145]. Their reaction mechanism was discussed for many years, but it seems evident, that the peroxide is homolytical cleaved and the ferric heme is converted to compound II (FeIVOH), (Fig. 4) [145, 146]. Allen oxide synthase (AOS) and hydroperoxide lyase (HPL) are only two out of this group, which are summarized as CYP74 enzymes. These enzymes do not form monohydroxides; rather rearrange hydroperoxides to different metabolites. Recently it was shown, that only single substitution changes AOS to HPL [146]. When 9-HPODE is transformed by AOS and HPL, a 9(10)epoxide and a carbon centered radical at C-11 are formed. Due to a bulky phenylalanine residue, AOS stabilizes the radical and favors formation of an allenoxide, whereas HPL with leucine in homologous position produces a hemiacetal following C-C scission between C-9 and C-10, and subsequently decays to aldehydes [146, 147]. Hydroperoxide isomerases transform hydroperoxides to diols. The reaction mechanism with suprafacial hydrogen abstraction and oxygenation is typical for P450. In fungi, only recently a cytochrome P450 reaction was identified in Aspergilli [148]. The PpoA (5,8-LDS) enzyme of A. nidulans was characterized as a fusion enzyme, which first dioxygenates 18:2n-6 to 8R-HPODE by a dioxygenase, and further isomerizes 8R-HPODE to 5,8DiHODE. Isomerization was obtained by a P450 related enzyme, as the gene sequence showed characteristic properties (e.g. cysteine heme ligand and EXXR motif) [148].. 22.

(227) Figure 4. Catalytic reaction cycle of cytochrome P450 and CYP74, according to Brash [145] with a few modifications.. 23.

(228) Aims. The first discovered LDS of G. graminis evoked interest due to the catalytic and structural similarities to PGHS. In order to study the enzyme, and to elucidate more similarities and differences, the overall objective was to invest a proper expression system for the 8-DOX of 7,8-LDS and to carry out mutational studies at the catalytic site. Specific objectives were to: • Develop an appropriate expression system for 7,8-LDS of G. graminis (papers I and II). • Determine the catalytically important tyrosine residue and the histidine heme ligand by site-directed mutagenesis (papers I and II). • Study different homologous residues to PGHS in 7,8-LDS by site-directed mutagenesis (papers I and II). In 2005, the genomes of A. fumigatus and familiar strains were published, and generated homologous protein sequences to PGHS and 7,8-LDS. Specific objectives were to: • Identify the fatty acid metabolites, formed from C16 to C20 fatty acids by mycelia of Aspergilli (paper III). • Relate specific dioxygenase activities to the gene sequences by studying mutant strains (paper III). • Determine the stereospecific hydrogen abstraction by 5,8LDS and by two novel dioxygenases, 8,11-LDS and 10DOX, using stereo specifically deuterated substrates (paper III). • Develop an HPLC method to elucidate the stereo chemistry of mono- and dihydroxy metabolites formed by LDS and DOX (paper V). • Clone and express 10-DOX of A. fumigatus (paper IV). • Determine structural differences between 8-DOX and 10DOX, and elucidate catalytically important residues for 10dioxygenase activity (paper IV).. 24.

(229) Methods. Expression systems In order to study enzymes, productions of large amounts of recombinant proteins are often needed. Many expression systems are established for directed protein synthesis of a foreign gene with distinct advantages and disadvantages. The probably most used cell line for expression is Escherichia coli. High recovery, little laboratory effort and low costs give this system the preference. However, no posttranslational modifications and formation of insoluble inclusion bodies require often other systems for expression. Insect cells, yeasts and mammalian cell lines are often utilized for production of proteins from eukaryotic organisms. In the present study, the yeast Pichia pastoris and insect cells were used to express 7,8-LDS and 10-DOX.. Expression in Pichia pastoris Pichia pastoris, the methylotrophic yeast, is an established and widely used heterologous expression system. Fatty acid metabolizing enzymes, like manganese lipoxygenase and the porcine leucocyte 12-LOX were successfully expressed in this yeast [49, 149]. P. pastoris exhibits a series of advantages. The yeast is characterized by a rapid growth rate, allowing high cell-density fermentation, which leads to high levels of heterologous proteins [150, 151]. In contrast to prokaryotic expression systems, the yeast can be used to generate eukaryotic and prokaryotic proteins without need of a complex medium. P. pastoris is known for its O- and N-linked glycosylation, which sometimes is necessary to produce proper folded, active, eukaryotic enzymes. On the other hand, extensive or different glycosylation might lead to inactive enzymes. However, the yeast can also be manipulated to humanize the glycosylation pattern [152-154]. Nowadays, a wide range of different Pichia strains are available. P. pastoris uses methanol as a carbon source and possesses several unique enzymes for the methanol utilization pathway [155]. The alcohol oxidase (AOX) is involved in the first step of methanol metabolism, and oxidizes methanol to formaldehyde and hydrogen peroxide, which is further metabolized to water and oxygen by the peroxisome catalase [155]. Since the AOX enzyme has a low oxygen affinity, the AOX promoter is up-regulated to 25.

(230) compensate AOX production. Therefore the AOX promoter, induced by methanol, can be used to drive expression of recombinant proteins [156]. Glycerol represses the promoter, and allows generation of biomass.. Expression vector Many commercial vectors are available. The pPICZ-vector (3,3 kb) was chosen to express 8-DOX of 7,8-LDS. As described above, an AOX1promoter drives the expression, induced by methanol. The Sh ble gene (Streptoalloteichus hindustanus bleomycin gene) allows the use of the antibiotic (Zeocin) as a selective marker. Hörnsten et al. sequenced and cloned the gene of 7,8-LDS [118]. Briefly, the gene of 5.5 kb was ligated into pGEM5Zf+, and the three introns were replaced by the corresponding cDNA fragment that was obtained by RT-PCR. The 5’- end was modified by introducing a SpeI site and a Kozak sequence (acgATGG). The stop codon was removed and an EcoRV site was inserted. The open reading frame (KpnIXbaI fragment) was ligated into pPICZB, in frame with a His-tag.. Transformation and expression of 8-DOX in P. pastoris The wild type P. pastoris strain X33 was used for transformation and expression. Electro competent cells were prepared as described in the instructions [157]. The construct was linearized by PmeI, purified, electroporated into P. pastoris, and selected on Zeocin containing YPDS plates. Selected transformants were grown in buffered glycerol complex medium to create biomass. Changing the medium to buffered methanol complex medium induces expression of 7,8-LDS. After 1-3 days, the cells were pelleted, washed, and stored at -80ºC. In order to confirm homologous recombination, genomic DNA of positive transformants was extracted, and PCR verified the incorporation of the gene.. Expression in insect cells The baculovirus based system Another well established, and during the last decades further developed, expression system represents the baculovirus induced expression in insect cells. Among many proteins, PGHS could be successfully expressed by this system [158]. Similar to P. pastoris, insect cells are able to produce eukaryotic proteins, including posttranslational modifications and processing. Furthermore, the virus can be propagated to high titer in insect cells, and facilitates growth in suspension cultures. The baculovirus is not contagious to vertebrates, and the promoter is inactive in most mammalian cells, which enables a safe working environment [159]. Another advantage is the size of 26.

(231) the viral genome that can harbor large fragments of foreign DNA. The cell line used for infection derives from the fall armyworm Spodoptera frugiperda (Sf9/Sf21). The common expression systems use the lytic Autographa california nuclear polyhedrosis virus (AcMNPV). Insect cells are coinfected by a transfer vector, containing the gene of interest and a strong polyhedrin promoter, and the baculovirus. During homologous recombination, the wild type virus loses the polyhedrin gene, and the foreign DNA will be integrated. The protein of interest is expressed by control of the remaining polyhedrin promoter. During the past decades this system was improved and changed by many companies. In the present study the “Bac-to-Bac” expression system was used (Fig. 5) [160]. It utilized the pFastBac vector, as a transfer vector. This procedure selects recombinant viruses before infection. The transfer vector was transformed into bacmid (baculovirus shuttle vector) containing Escherichia coli cells (DH10 Bac). The foreign gene was integrated by transposition. Blue-white screening and antibiotic selection identified positive clones, the recombinant bacmid DNA was extracted, and used for transfection of insect cells. The infected cells released recombinant baculoviruses into the medium, which could be amplified and used for further transfection and final expression.. Figure 5. Overview of the “Bac-to-Bac” expression system. 27.

(232) The plasmid based expression system Even though the baculovirus based expression is established, it displays a method, which is time consuming and needs a high laboratory effort. Invitrogen presents a quick method that enables screening of expressed proteins, named InsectSelect [161]. This system utilizes the vector pIZ/V5-His to express the gene of interest. In the present study, the open reading frames of 8-DOX, 7,8-LDS and 10-DOX were cloned into the transfer vector, yielding pIZ/V5-His_8-DOX, pIZ/V5-His_7,8-LDS, and pIZ/V5-His_10DOX (Fig. 6). The OpIE2 promoter, from the Orgyia pseudotsugata multicapsid nucleo polyhedrosis virus, drives the expression of the foreign protein. For expression of 10-DOX, an additional construct, with retention of the native stop codon was prepared (pIZ_10-DOX). In contrast to the baculovirus based system, the plasmid will be introduced directly into insect cells by liposomes. Three days post transfection, cells can be harvested and the recombinant protein can be used for further investigations. The major drawbacks are relative low levels of protein outcome, and high expenses for liposomes. Nevertheless, this method could be used to screen many mutants for activity.. Figure 6. Vector graphs of 8-DOX, 7,8-LDS and 10-DOX for plasmid driven expression in insect cells. The C-terminal domain of 10-DOX lacks the characteristic cysteine residue of P450.. 28.

(233) Enzyme activity assay Cells of P. pastoris were washed with water and suspended in phosphate buffer (pH 7.4). After disruption with glass beads [162], cell debris was spun down, and the supernatant was used to purify the recombinant enzyme, or for activity tests. Insect cells were lysed in Tris-HCl buffer (0.04% Tween 20; 5% glycerol; 1mM glutathione; 0.5M NaCl; protease inhibitor, Complete-EDTA free; pH 7.4), and sonicated. Cell debris was spun down and the supernatant was used for enzyme activity. The crude supernatant from both systems was incubated with 50-100μM linoleic acid, which presents the main substrate for 7,8-LDS and 10-DOX, for 30 minutes on ice. The reaction was terminated by 3-5 volumes of ethanol, and the metabolites were extracted by solid phase extraction on a C18 column. In some experiments an internal standard was added ([2H4]13HODE). Finally, the metabolites were analyzed by LC-MS/MS.. Site-directed mutagenesis Site-directed mutagenesis was performed using the QuikChange mutagenesis strategy. This PCR technology utilizes Pfu DNA polymerase and 42 to 46 bp primers, containing the desired mutation. Forward and reverse reactions were mixed in separate tubes. After 3 cycles the reactions were combined in order to run additional 16 cycles [163]. The parental plasmid was digested by the restriction enzyme DpnI, which specifically cleaves methylated DNA. Transformation of E. coli was used to amplify the mutated plasmid. Mutations were confirmed either by restriction analysis, or by sequencing.. Western blot analysis Western blot analysis was utilized to confirm protein expression. This was of fundamental importance for enzymes that did not show activity. SDS-PAGEs (sodium dodecyl sulfate polyacrylamid gel electrophoresis) were performed on 8% polyacrylamide gels, suitable for 7,8-LDS, 10-DOX and their mutants. After separation of the proteins by their size, they were blotted onto nitrocellulose membranes. The recombinant enzymes could be probed by an anti-His antibody, directed against a C-terminal polyhistidinetag. The secondary antibody, coupled to horseradish peroxidase allowed detection by chemoluminescence.. 29.

(234) Synthesis of racemic hydroxy- and dihydroxy fatty acids In order to analyze the stereo specificity of the metabolites, enantiomeric standards are needed. Secondary alcohols can be oxidized to the corresponding ketone by a periodinane, called Dess-Martin reagent [164]. A racemic mixture is achieved when those oxidized metabolites are further reduced to the alcohol by sodium borohydride. The reaction was performed in chloroform for 40 min, and terminated by ethyl acetate. After the organic phase was washed and evaporated, the oxidized metabolites were dissolved in methanol, and immediately reduced to the racemic alcohols. This procedure can be also applied to form a mixture of diastereomers of dihydroxy fatty acids. Additional, in order to identify the stereochemistry of diastereomers, authentic standards with known configuration are needed. 8R-HODE was extracted from G. graminis and the S stereoisomer of 10-HODE was formed by Lentinula edodes [44, 165]. Agaricus bisporus converts 18:2n-6 to 8R,11S-DiHODE [42]. 5S,8R-DiHODE, with known stereochemistry, was formed by A. nidulans [166].. High performance liquid chromatography High performance liquid chromatography (HPLC) is a wide-spread analytical method to separate a mixture of compounds. The choice of the right column depends on the compounds affinity to the mobile and stationary phase. In the present study dioxygenated fatty acids and further metabolites were analyzed. Mono- and dihydroxy fatty acids and hydroperoxy fatty acids could be separated on C18 columns, eluted by methanol/water/acetic acid (80/20/0.01 or 75/25/0.01), in an isocratic mode. In order to separate and identify diastereomers of dihydroxy fatty acid, separation was performed on silica normal phase columns. As a mobile phase, hexane/isopropanol/acetic acid (95/5/0.01) was used at a flow of 0.3-0.6 ml/min. To achieve separation of enantiomers of monohydroxy fatty acids, chiral columns are needed. Separation of 8R/S-HODE was carried out on a Chiralcel OB-H column when eluted with hexane/isopropanol/acetic acid (95/5/0.01 or 92/8/0.05). For analysis of 10R/S-HODE, the Reprosil Chiral NR column was used and eluted by hexane/isopropanol/acetic acid (97/3/0.01). This column has an aromatic ring as a chiral selector, and is also usable for chiral analysis of hydroperoxy fatty acids [167].. 30.

(235) Mass spectrometry This method separates compounds by their mass-to-charge ratio (m/z), which differs to other spectroscopic methods. In the mass spectrometer, the molecules break into smaller fragments, which are characteristic and gives the compound a unique fingerprint. Every mass spectrometer consists of an ion source, an analyzer and a detector. In order to analyze fatty acid metabolites a linear ion trap mass spectrometer (LTQ, Thermo Fisher) was applied. In this instrument, ions are generated by electrospray ionization (ESI). The sample enters the ESI nozzle, which has a positive or a negative voltage, depending of the charge of interest. Fatty acids are commonly analyzed as negative ions. Afterwards, when the solvent of the charged droplets evaporates, their size decrease until the Coulombic forces exceed, and smaller charged droplets emerge. This process continues, until individually charged analytes are formed. The ions travel through the ion transfer tube into the analyzer, a linear quadrupole ion trap. The instrument is now able to trap all ions, to monitor single ions, or to excite and fragment precursor ions. The latter can be repeated in order to specify the spectrum for a compound. Finally, the ions are ejected and monitored by the detector. The mass spectrometer is able to analyze ions simultaneously. However, a preseparation by a coupled HPLC column was performed, and allowed additional identification by retention time. When using a hexane/isopropanol/acetic acid mobile phase, the effluent was combined in a Tjunction with isopropanol/water (3/2; 0.2-0.3 ml/min), and further subjected to electrospray ionization (ESI).. 31.

(236) Results. Recombinant 7,8-LDS Recombinant 7,8-LDS has dual enzyme activities and transforms linoleic acid sequentially to 8R-HPODE and to 7S,8S-DiHODE. Native LDS has stable dioxygenase activity. However, the hydroperoxide isomerase activity was highly variable, occasionally undetectable directly after purification, but it could be resorted by storage on ice [117]. The present study focused on the mechanism of 8R-DOX in comparison with PGHS. The first expression constructs lacked some C-terminal amino acids due to a sequencing error in the original publication [118], and yielded prominent the 8R-DOX activity, usually with little interfering hydroperoxide isomerase activity when expressed in insect cells. Expressed in P. pastoris, we noted significant formation of 5,8R-DiHODE and 8R,13-DiHODE (paper I). The latter was likely formed from 8,9-epoxyoctadecadienoic acid by hydrolysis. The aberrant formation of 5,8-DiHODE led us to investigate the 5,8-LDS of A. nidulans (paper I), and to collaborate with Dr. Keller from the University of Wisconsin, and Dr. Hamberg from the Karolinska Institute, Stockholm (paper III). Expression of 7,8-LDS in insect cells with the corrected expression construct (1165 amino acids, GenBank Accession No AF124979 ) yielded prominent 8R-DOX and hydroperoxide isomerase activity, as summarized in Fig. 7. As expected, the major products were 8R-HPODE and 7S,8S-DiHODE, but small amounts of 6S,8R-DiHODE and 8R,11S-DiHODE were also formed in analogy with 7,8-LDS of Magnaporthe oryzae (Jernerén, unpublished observation). For analysis of the 8R-DOX activity and its relation to PGHS, it was advantageous to use the construct with little interfering hydroperoxide isomerase activity. For this purpose, the plasmid-driven expression in insect cells was rapid, robust and convenient. The baculovirus expression system was also evaluated, but it offered only neglectable advantage.. Site-directed mutagenesis of 8-DOX (papers I and II) Identification of the catalytically important tyrosine of 7,8-LDS was the first objective. Tyr376, the homologous residue of Tyr385 of PGHS, was replaced by phenylalanine. The mutant did not show dioxygenase activity. However, Tyr376Phe, expressed in P. pastoris, transformed 8R-HPODE to 5,8-DiHODE. 32.

(237) Figure 7. Metabolites formed by recombinant 7,8-LDS.. The heme ligand histidine of PGHS (His388) was conserved in the motif YRWH379 of 7,8-LDS. His379 was mutated to glutamine, and resulted in an inactive enzyme. Both mutation, Tyr376Phe and His379Gln, were repeated in insect cells with the same outcome. The proposed distal histidine (His203) was replaced by glutamine. His203Gln, expressed in P. pastoris and by the plasmid driven expression in Sf21 yielded an inactive enzyme. However, expression by baculovirus increased the protein level, and His203Gln converted 18:2n-6 to detectable 8R-HPODE. In order to carry out investigations at sites of posttranslational modification, a glycosylation site (N216LT) was disrupted by replacing asparagine 216 by glutamine. The P. pastoris expressed mutant showed no significant enzyme activity. Tyr348 of PGHS is known to be important for positioning of the substrate at the active site [85]. The homologous tyrosine 329 of 7,8-LDS was mutated to phenylalanine, which retained low 8R-DOX activity. However, Tyr329Leu abolished enzyme activity. Tryptophan 378, conserved in the YRW378H motif, was mutated to phenylalanine and serine, and both mutants did not transform linoleic acid. Furthermore, Tyr531 was mutated to phenylalanine, and this replacement resulted in an inactive 8-dioxygenase. Another objective was to identify the substrate binding residue. The proposed lysine 540 was replaced by leucine, glutamine and the charged amino acid arginine. All three mutants did not transform 18:2n-6 to 8-HPODE. Site-directed mutagenesis studies are summarized in Table 1. The successful expression of all mutants was confirmed by Western blot analysis. 33.

(238) Table 1. Overview of site-directed mutagenesis studies at the dioxygenase site of 7,8-LDS.. 7,8-LDS. r-LDS His203Gln Asn216Gln Tyr329Phe Tyr329Leu Tyr376Phe Trp378Phe Trp378Ser His379Gln Tyr531Phe Lys540Leu Lys540Gln Lys540Arg a. Expres- Baculovirus sion in P. based expastoris pression + + + + + + + + + -. Plasmid driven expression + + + + + + + + + + + +. 8R-DOX activity +++ +a + -. Activity could be detected, when expressed by baculovirus in Sf21.. Dioxygenases in Aspergilli (paper III) Mycelia of A. fumigatus convert linoleic acid to several mono- and dihydroxy fatty acids, which were analyzed by LC-MS/MS. The two major monohydro(pero)xy fatty acids were identified as 8-H(P)ODE and 10H(P)ODE. CP-HPLC recognized both metabolites as R stereoisomers (described below). Furthermore, dihydroxy fatty acids were detected, and identified as 5,8-DiHODE and 8,11-DiHODE. Both were formed from 8HPODE. NP-HPLC recognized the diastereoisomers as 5S,8R- and 8R,11SDiHODE. Cell-free preparations of mycelia (nitrogen powder) converted linoleic acid to additional metabolites, analyzed as 10-oxy-8E-decenoic acid (10-ODA) and 10-ketooctadecadienoic acid (10-KODE). These oxylipins are presumably formed from 10-HPODE during incubation, and 10-KODE by dehydration in the heated transfer system of the mass spectrometer. Surprisingly, the profile of oxylipins varied when the growth conditions were changed. Mycelia, growing at 37ºC, converted 18:2n-6 mainly to 8RHODE. When the growth temperature was decreased to 22ºC, the formation of 10R-HODE was increased. The transformation to 10R-HODE could be even more elevated, when the growth length was extended at 22ºC. The temperature had also influence of the formation of dihydroxy metabolites. The conversion to 8R,11S-DiHODE could be increased at a growth temperature of 22ºC. This suggested differential expression of the enzymes (5,8-LDS, 8,11-LDS, and 10-DOX). 34.

(239) Catalytic mechanism (paper III) Incubation of cell free preparations of A. fumigatus (Fresen.) with 8RHPODE increased formation of 5S,8R-DiHODE and 8R,11S-DiHODE. However, the biosynthesis of 10R-HODE was not elevated. This fact recommends a distinct pathway to form 10R-HODE. In order to identify the reaction mechanism of 8-DOX and 10-DOX, stereospecifically deuterated substrates were applied. Incubation with [(8R)2 H]18:2n-6 retained the pro-R hydrogen in 8R-HODE, 10R-HODE, 5S,8RDiHODE, and 8R,11S-DiHODE. That implies pro-S hydrogen abstraction, and antarafacial oxygen insertion at C-8 and C-10 (Fig. 8). Furthermore, A. fumigatus converts [(11S)-2H]18:2n-6 to 8R,11S-DiHODE with loss of the deuterium label, which suggests pro-S hydrogen abstraction and suprafacial hydroxylation at C-11. This could be confirmed by incubation with [(11R)2 H]18:2n-6, and retention of the deuterium label. A. fumigatus oxidized [(5S)-2H]18:2n-6 to 5S,8R-DiHODE with retention of the deuterium, which suggests pro-S hydrogen abstraction of 8R-HPODE, and suprafacial hydroxylation.. Figure 8. Mechanism of dioxygenases and isomerases of A. fumigatus.. Identification of dioxygenases (paper III) After the genomes of A. fumigatus and A. nidulans were published, in each strain three genes with homology to 7,8-LDS were identified (ppoA, ppoB, ppoC). The aim was to relate special fatty acid metabolites to the enzyme, coded by a certain homologous gene. Knockout strains from A. fumigatus 35.

(240) (Af293) were applied to identify enzyme activity. Deletion of ppoA abolished formation of 5S,8R-DiHODE, but had no influence on formation of 10R-HODE. The ppoC knockout strain showed no significant formation of 10RHODE, and formed 8R-HODE and 5S,8R-DiHODE as the wild type strain. Deletion of ppoB did not show an alteration in the oxylipin profile, formed from 18:2n-6, however, the wild type strain did not form 8,11-DiHODE. Gene deletion studies carried out in A. nidulans resulted in the same outcome, as deletion of ppoA abolished formation of 5,8-DiHODE and the "ppoC strain did not generate 10-HODE. It seems likely that ppoA and ppoC of A. fumigatus and A. nidulans code for dioxygenases, which form 8RHODE and 5S,8R-DiHODE, and 10R-HODE, respectively.. Stereochemistry of mono- and dihydroxy metabolites (paper IV) It is of fundamental importance to identify the stereochemistry of metabolites, when mechanistic studies on enzymes are performed. Enantiomers possess identical chemical and physical properties, and are difficult to separate. Chiral HPLC columns are used to separate a pair of enantiomers. Furthermore, authentic standards are needed. In the present study, the stereochemistry of 8-HODE and 10-HODE, formed by Aspergilli, was identified. 8R-HODE formed by G. graminis and 10S-HODE produced by Lentinula edodes were utilized as authentic standards [44, 165, 168]. As described, Dess-Martin reagent was used to yield racemates. The enantiomers of 8HODE could be separated on a cellulose tribenzoate coated silica column (Chiralcel OB-H), and the R stereoisomer eluted after the S isomer. The reconstructed ion chromatogram of 8-HODE, formed by A. fumigatus, showed the R isomer to 97%. Racemic 10-HODE could be separated on Reprosil Chiral NR column, and with aid of the S isomer standard of L. edodes, the elution order could be identified. 10S-HODE eluted before its R stereoisomer. A. fumigatus and A. nidulans formed 10R-HODE to 88% and 77%, respectively. In order to identify the stereochemistry of dihydroxy fatty acids, diastereomers were separated by NP-HPLC. 8R,11S-DiHODE with known configuration was extracted from A. bisporus [42], and used as an authentic standard. The diastereomers of [8,11-2H2]8,11-DiHODE, produced by the Dess-Martin reagent and further reduction, were dissolved on NP-HPLC. The second eluting compound had R/S or S/R configuration, since it showed same retention time as the standard 8R,11S-DiHODE. 8,11-DiHODE formed by A. fumigatus eluted at the same retention time as 8R,11S-DiHODE. It seemed likely, that this metabolite is an R/S isomer, as it derives from 8R-HPODE. 36.

(241) The diastereomers of racemic [5,8-2H2]5,8-DiHODE were also separated by NP-HPLC. The R/S and S/R diastereomers eluted prior metabolites with S/S and R/R configuration, since the 5S,8R-DiHODE, formed by A. nidulans and with known configuration [166], eluted with the first diastereomer. 5,8DiHODE, formed by A. fumigatus, showed identical configuration (S/R), as it eluted at the same retention time and derives from 8R-HPODE. The results are summarized in Table 2. Table 2. Summary of stereochemical analysis of metabolites formed from 18:2n-6 by A. fumigatus. Metabolite 8-HODE. 10-HODE 5,8-DiHODE 8,11-DiHODE a. Column Chiralcel OB-H cellulose tribenzoate coated silica Reprosil Chiral NRa NP-HPLC silica column NP-HPLC silica column. SR. A. fumigatus 8R-HODE. SR. 10R-HODE. RS; SRSS; RR. 5S,8R-DiHODE. SS; RR RS; SR. 8R,11S-DiHODE. Elution order. The selector of Reprosil Chiral NR has not been disclosed.. Cloning and expression of 10-DOX (paper V) 10-DOX of A. fumigatus was cloned by RT-PCR. The cDNA was sequenced, and a comparison with the predicted gene sequence confirmed 13 exons and 12 short introns. The open reading frame (3366 bp) was ligated into the pIZ/V5-His vector, in order to express 10-DOX by the plasmid driven insect cell system. For expression, two different constructs were used. One consists of a native stop codon (pIZ_10DOX). In the other construct, the stop codon was removed, and the sequence was extended by 34 amino acids, containing a C-terminal V5 epitope and a His6 tag (pIZ/V5His_10DOX). Both recombinant 10-DOX transformed linoleic acid to 10R-HODE (~90%) and to 8R-HODE (~10%) (Fig. 9), which is in line with the native enzyme. The expression could be confirmed by Western blot analysis. Incubation with [(8R)-2H]18:2n-6 showed pro-S hydrogen abstraction at C-8, since the deuterium label was retained in the metabolites. Furthermore, two side products were detected and identified as 10-ODA (10 oxydecenoic acid) and an epoxy alcohol (~1%), (Fig. 9). Albeit the amount of the epoxyalcohol was low, the formation raised interest. MS/MS analysis of this compound elucidated the final structure as 12(13)-epoxy10R-hydroxy-18:1. NP-HPLC separated the diastereomers of this compound, and showed stereospecific formation of the epoxy alcohol. The absolute configuration was identified by using vernolic acid as a substrate. Minus 37.

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