| 1. |
- Wennman, Anneli
(författare)
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The structural basis for the catalytic specificity of manganese lipoxygenases : 3D structure analysis of the lipoxygenase of Magnaporthe oryzae
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipoxygenases (LOX) catalyze regio- and stereospecific oxygenation of polyunsaturated fatty acids to hydroperoxides. These hydroperoxides are further metabolized to leukotrienes and lipoxins in mammals, and are involved in asthma and inflammation. LOX of animals and plants contain iron as catalytic metal (FeLOX). Filamentous fungi use both FeLOX, and manganese containing LOX (MnLOX). The role of LOX in fungi is still not known. This thesis focuses on expression of novel MnLOX, analyses of their reaction mechanism and products by HPLC-MS/MS, protein crystallization and analysis of the first MnLOX structure. MnLOX from G. graminis, M. salvinii, M. oryzae, F. oxysporum and C. gloeosporioides were expressed in Pichia pastoris, purified and characterized by HPLC-MS/MS. All MnLOX catalyzes suprafacial hydrogen abstraction and oxygen insertion. Replacement of one Ile to Phe in the active site of MnLOX of G. graminis could switch the mechanism from suprafacial to mainly antarafacial. MnLOX of F. oxysporum was interesting since it catalyzes oxygenation of linoleic acid to 11R- instead of the more common 11S-hydroperoxides. This feature could be attributed to a single Ser/Phe exchange in the active site. We found that Gg-MnLOX utilizes hydrogen tunneling in the reaction mechanism, but was slightly more temperature dependent than soybean FeLOX. It is an intriguing question why some fungal LOX use manganese and not iron as catalytic metal and whether the large redox potential of Mn2+/Mn3+ (1.5 V) can be tuned close to that of Fe2+/Fe3+ (0.77 V) for redox cycling and catalysis.We present crystallization conditions for two MnLOX, and the 2.07 Å crystal structure of MnLOX from M. oryzae, solved using sulfur and manganese single anomalous dispersion (SAD). The structure reveals a similar metal coordinating sphere as FeLOX but the metal ligand Asn473 was positioned on a short loop instead of a helix and formed interactions with a conserved Gln. This feature could be essential for the use of manganese as catalytic metal in LOX. We found three Phe residues that likely facilitate the suprafacial hydrogen abstraction and oxygen insertion for MnLOX.These findings provide new insight into the unique reaction mechanism of MnLOX.
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| 2. |
- Cristea, Mirela, 1976-
(författare)
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Expression of Manganese Lipoxygenase and Site-Directed Mutagenesis of Catalytically Important Amino Acids : Studies on Fatty Acid Dioxygenases
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.
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| 3. |
- Hoffmann, Inga, 1984-
(författare)
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Discovery of Novel Fatty Acid Dioxygenases and Cytochromes P450 : Mechanisms of Oxylipin Biosynthesis in Pathogenic Fungi
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dioxygenase-cytochrome P450 (DOX-CYP) fusion enzymes are present in diverse human and plant pathogenic fungi. They oxygenate fatty acids to lipid mediators which have regulatory functions in fungal development and toxin production. These enzymes catalyze the formation of fatty acid hydroperoxides which are subsequently converted by the P450 activities or reduced to the corresponding alcohols. The N-terminal DOX domains show catalytic and structural homology to mammalian cyclooxygenases, which belong to the most thoroughly studied human enzymes.7,8-Linoleate diol synthase (LDS) of the plant pathogenic fungus Gaeumannomyces graminis was the first characterized member of the DOX-CYP fusion enzyme family. It catalyzes the conversion of linoleic acid to 8R-hydroperoxylinoleic acid (HPODE) and subsequently to 7S,8S-dihydroxylinoleic acid by its DOX and P450 domains, respectively. By now, several enzymes with homology to 7,8-LDS have been identified in important fungi, e.g., psi factor-producing oxygenase (ppo)A, ppoB, and ppoC, of Aspergillus nidulans and A. fumigatus.By cloning and recombinant expression, ppoA of A. fumigatus was identified as 5,8-LDS. Partial expression of the 8R-DOX domains of 5,8-LDS of A. fumigatus and 7,8-LDS of G. graminis yielded active protein which demonstrates that the DOX activities of LDS are independent of their P450 domains. The latter domains were shown to contain a conserved motif with catalytically important amide residues. As judged by site-directed mutagenesis studies, 5,8- and 7,8-LDS seem to facilitate heterolytic cleavage of the oxygen-oxygen bond of 8R-HPODE by aid of a glutamine and an asparagine residue, respectively.Cloning and expression of putative DOX-CYP fusion proteins of A. terreus and Fusarium oxysporum led to the discovery of novel enzyme activities, e.g., linoleate 9S-DOX and two allene oxide synthases (AOS), specific for 9R- and 9S-HPODE, respectively. The fungal AOS are present in the P450 domains of two DOX-CYP fusion enzymes and show higher sequence homology to LDS than to plant AOS and constitute therefore a novel class of AOS.In summary, this thesis describes the discovery of novel fatty acid oxygenases of human and plant pathogenic fungi and the characterization of their reaction mechanisms.
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| 4. |
- Nilsson, Tomas, 1977-
(författare)
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Mass Spectrometric Analysis of Oxylipins : Application to Cytochrome P450-Dependent Metabolism
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. In contrast, CYP4F8 metabolizes an epoxyalcohol derived from 12R-HPETE, 11R,12R-epoxy-10-hydroxyeicosatrienoic acid, to the ω3 hydroxy metabolite. Conclusively, it was demonstrated that LC-MS/MS could be used for the analysis and separation of hydroperoxides and epoxyalcohols for metabolic studies.
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| 5. |
- Stark, Katarina, 1975-
(författare)
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Catalytic Properties and Tissue Distribution of Cytochrome P450 4F8 and 4F12 : Expression of CYP4F8 in Eye Tissues and Psoriatic Lesions
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human cytochrome P450 (CYP) family of monooxygenases is important for metabolism of drugs and endogenous compounds, e.g., vitamin A and D, cholesterol, steroids, fatty acids, and eicosanoids. This thesis describes the tissue distribution, catalytic properties, and possible function of CYP4F8 and CYP4F12. To this respect, methods for immunohistological analysis, and real-time PCR for analysis of their transcripts, were developed.CYP4F8 was originally cloned from human seminal vesicles and proposed to catalyze 19-hydroxylation of prostaglandin H2 (PGH2). This notion could now be supported, as cyclooxygenase-2, CYP4F8, and microsomal prostaglandin E synthase-1 were found to be co-localized in the epithelial linings of seminal vesicles. The three enzymes were also co-localized in the suprabasal layers of epidermis, suggesting a similar function of CYP4F8 in skin. Real-time PCR showed that CYP4F8 mRNA was more than 10-fold increased in psoriatic lesions compared to non-lesional skin. CYP4F8 immunoreactivity was also found in kidney cortex, transitional epithelium, corneal epithelium, and retina. Although transcripts of all three enzymes were detectable in retina, no co-localization was found. Pro inflammatory stimuli were found to increase CYP4F8 mRNA expression in cultured epidermal and corneal keratinocytes. In these tissues CYP4F8 might oxidize fatty acids or other eicosanoids than PGH2.CYP4F12 was originally cloned from the liver and small intestine, and found to oxidize arachidonic acid and two anti-histamines. Immunohistological studies showed that CYP4F12 immunoreactivity was present mainly in the gastrointestinal tract, e.g., stomach, ilium, and colon, but also in placenta. Although CYP4F8 and CYP4F12 have catalytic properties in common, there are important differences. CYP4F12 does not oxidize PGH2, certain eicosanoids, and fatty acids. The prominent expression in the gut suggests that CYP4F12 might be involved in oxidation of drugs.
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| 6. |
- Wigren, Jane, 1967-
(författare)
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Identification of natural activators of the nuclear receptor peroxisome proliferator-activated receptor : relevance to the pathogenesis of atherosclerosis
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Polyunsaturated fatty acids induce peroxisome proliferation. This phenomenon is mediated by the ligand-dependent transcription factor peroxisome proliferator-activated receptor (PPAR). This thesis is an investigation on the role of eicosanoids and oxidized products of linoleic acid for the activation of PPARs. Special emphasis was given to the subtype PPAR/gamma/ in the context of atherosclerosis.It had earlier been shown that arachidonic acid induces peroxisome proliferation in Morris Hepatoma 7800C1 cells. We investigated whether this effect could be attributed to a cytochrome P-450IVA product of arachidonic acid, 20-hydroxy-arachidonic acid. Arachidonic acid, but not 20-hydroxy-arachidonic acid induced lauryl-CoA oxidase activity. The effect of arachidonic acid was potentiated by all-trans retinoic acid, consistent with the notion that PPAR/RXR heterodimers mediate the effect.Several reports in the litterature were suggestive of an important role of peroxisomes in eicosanoid metabolism. However, nobody had isolated pure peroxisomes and investigated their eicosanoid metabolizing ability. We therefore investigated the ability of peroxisomes to metabolize the eicosanoid 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). Incubation of tritium-labeled 12(S)-HETE with isolated peroxisomes from rat liver or kidney peroxisomes demonstrated that more than 90 % of the diethyl ether extractable radioactivity was due to a single metabolite, identified as 8-hydroxy-6, 8, 12-octadecatrienoic acid (8-OH-16:3). This compound was apparently formed by two rounds of ß-oxidation. The data for the first time provided conclusive evidence for a role of peroxisomes in HETE metabolism.The second half of the thesis deals with the identification of natural PPAR/gamma/ ligands in LDL from atherosclerotic patients and in activated macrophages. Analyses of the endogenous content of selected monohydroxy fatty acids in LDL isolated from a group of patients diagnosed with intermittent claudication, showed the presence of 9- and 13-HODE, 5-, 12-, and 15-HETE. These compounds activated PPAR/gamma/ in macrophages and preferentially recruited the coactivator protein CBP to PPAR/gamma/RXR/alpha/ heterodimers. 15-deoxy-/DELTA/12,14-Prostaglandin J2 (15-deoxy-/DELTA/12,14-PGJ2) was identified as a PGD2 metabolite in macrophage cultures (see below). It induced the interaction of PPAR/gamma/RXR/alpha/ heterodimers with both CBP and SRC-1. This observation suggests that different PPAR/gamma/ ligands may induce different effects through a single kind of receptor by differential recruitment of coactivators.Although PGD2, is not a PPAR/gamma/ ligand, it induces PPAR/gamma/-mediated effects in IFN-/gamma/-stimulated RAW 264.7 macrophages, suggesting that the effects required metabolism. We therefore investigated PGD2 metabolism in macrophage cultures, and determined the capacity of these metabolites to activate PPAR/gamma/. Two novel (/DELTA/12-PGD2, 15-deoxy-/DELTA/12,14-PGD2) and two previously known PPAR/gamma/ activators (/DELTA/12-PGJ2 and 15-deoxy-/DELTA/12,14-PGJ2) were identified by mass spectrometry. The structural difference between the novel products and the previously recognized PPAR/gamma/ agonists , /DELTA/12-PGJ2 and 15-deoxy-/DELTA/12,14-PGJ2, is that they contain a 9/alpha/-hydroxy group and lack a /DELTA/9,10 double bond. Two novel PPAR/gamma/ activators were formed in equal or greater amounts and were more potent activators of PPAR/gamma/ in macrophages.
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