Biosynthesis of allene oxides in Physcomitrella patens

Background: The moss Physcomitrella patens contains C-18- as well as C-20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results: Our results show that PpAOS1 is highly active with both C-18 and C-20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C-20-substrates, exhibiting trace activity (similar to 1000-fold lower k(cat)/K-M) with C-18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. Conclusions: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C-18- and C-20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes

A unified viscoplastic model for high temperature low cycle fatigue of service-aged P91 steel

The finite element (FE) implementation of a hyperbolic sine unified cyclic viscoplasticity model is presented. The hyperbolic sine flow rule facilitates the identification of strain-rate independent material parameters for high temperature applications. This is important for the thermo-mechanical fatigue of power plants where a significant stress range is experienced during operational cycles and at stress concentration features, such as welds and branched connections. The material model is successfully applied to the characterisation of the high temperature low cycle fatigue behavior of a service-aged P91 material, including isotropic (cyclic) softening and nonlinear kinematic hardening effects, across a range of temperatures and strain-rates

Biochemical Characterisation of PpoA from Aspergillus nidulans

In der vorliegenden Arbeit wurden Psi-Faktor produzierende Oxygenasen, sogenannte Ppo-Enzyme, charakterisiert, die eine regulatorische Funktion für die Entwicklung und Mycotoxinproduktion in A. nidulans besitzen. Bioinformatische Analysen zeigten, dass Ppo-Enzyme im N-terminalen Sequenzabschnitt eine hohe Homologie zu Fettsäure-Häm-Dioxygenasen/Peroxidasen aufweisen, während der C-terminale Sequenzabschnitt den Cytochrom-P450-Enzymen ähnelt. Um die biochemischen Eigenschaften von PpoA und PpoC zu untersuchen, wurde ein heterologes Expressionssystem in E. coli und ein Proteinreinigungsprotokoll etabliert. Während PpoA sich mit großen Proteinausbeuten stabil bis zur Homogenität reinigen ließ, war PpoC in gereinigter Form instabil und verlor die enzymatische Aktivität innerhalb weniger Minuten. PpoA wurde als homotetrameres Häm-Protein identifiziert, das die Oxidation von einfach und mehrfach ungesättigten Fettsäuren katalysiert. Auf der Basis von Aminosäuresequenzvergleichen, einem charakteristischen Häm-CO-Spektrum und ESR-Spektroskopie-Analysen konnte die Cytochrom-P450-Domäne nachgewiesen werden. Untersuchungen des Reaktionsmechanismus zeigten, dass PpoA zwei verschiedene Häm-bindende Domänen verwendet, um zwei voneinander getrennt ablaufende Reaktionen zu katalysieren. In der Fettsäure-Häm-Dioxygenase/Peroxidase-Domäne wird Linolsäure zu 8R-HPODE oxidiert, indem in einem initialen Reaktionsschritt ein Tyrosyl-Radikal gebildet wird, welches ein Wasserstoffatom vom C-8 der Fettsäure abspaltet. Es entsteht ein kohlenstoffzentriertes Radikal, welches im folgenden Schritt mit molekularem Sauerstoff reagiert und 8R-HPODE bildet. Das so intermediär gebildete Zwischenprodukt wird in der Cytochrom-P450-Domäne anschließend zu 5,8-DiHODE isomerisiert. Im Gegensatz zu PpoA scheint PpoC nur eine N-terminale Fettsäure-Häm-Dioxygenase/Peroxidase-Domäne zu besitzen und katalysierte hauptsächlich die Oxidation von Linolsäure zu 10-HPODE und die weitere Reduktion zu 10-HODE. Es konnte keine Isomerase-Aktivität nachgewiesen werden. Allerdings ist 10-HPODE instabil und wurde entweder zu 10-KODE oxidiert oder zerfiel zu 10-ODA und volatilen C8-Körpern, die unter anderem für den charakteristischen Pilzgeruch verantwortlich sind. Mutagenese-Studien wiesen darauf hin, dass PpoC wahrscheinlich einen ähnlichen Mechanismus für die Oxidation von Linolsäure verwendet wie PpoA und hierbei auch ein Tyrosyl-Radikal für die Abspaltung eines Wasserstoffatoms vom Fettsäurerückgrat gebildet wird. Es konnte gezeigt werden, dass die unterschiedlichen Positionen der Oxidation am Fettsäurerückgrat durch Aminosäurereste in der Fettsäure-Häm-Dioxygenase/Peroxidase-Domäne beeinflusst werden. Diese schirmen entweder das C-8 oder C-10 der Fettsäure vor dem Angriff von molekularem Sauerstof sterisch ab.

PpoC from Aspergillus nidulans is a Fusion Protein with one active Heme

International audienceIn Aspergillus nidulans psi factor producing oxygenases (Ppos) are required for the production of so-called psi factors (precocious sexual inducer) - compounds that control balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different heme containing domains are predicted: a fatty acid heme peroxidase/dioxygenase domain for the N-terminal region and a P450 heme thiolate domain for the C-terminal region. While PpoA was shown to use both heme domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerisation), we found that PpoC apparently only harbours a functional heme peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyzes mainly the dioxygenation of linoleic acid (18:2Δ9Z,12Z), yielding 10-hydroperoxy linoleic acid (10-HPODE). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-keto linoleic acid (10-KODE) and 10-hydroxy linoleic acid (10-HODE). In parallel, decomposition of 10-HPODE into 10-oxo decenoic acid (10-ODA) and volatile C8 alcohols that are inter alia responsible for the characteristic mushroom flavour. Beside these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE to the respective epoxy alcohols: 12,13-Epoxy-8-hydroxy octadecenoic acid and 12,13-epoxy-10-hydroxy octadecenoic acid. By using site directed mutagenesis we could demonstrate that both enzymes share a similar mechanism for the oxidation of 18:2Δ9Z,12Z: They both use a conserved tyrosine for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain

Functional Characterization of CYP94-Genes and Identification of a Novel Jasmonate Catabolite in Flowers.

Over the past decades much research focused on the biosynthesis of the plant hormone jasmonyl-isoleucine (JA-Ile). While many details about its biosynthetic pathway as well about its physiological function are established nowadays, knowledge about its catabolic fate is still scarce. Only recently, the hormonal inactivation mechanisms became a stronger research focus. Two major pathways have been proposed to inactivate JA-Ile: i) The cleavage of the jasmonyl-residue from the isoleucine moiety, a reaction that is catalyzed by specific amido-hydrolases, or ii), the sequential oxidation of the ω-end of the pentenyl side-chain. This reaction is catalyzed by specific members of the cytochrome P450 (CYP) subfamily CYP94: CYP94B1, CYP94B3 and CYP94C1. In the present study, we further investigated the oxidative fate of JA-Ile by expanding the analysis on Arabidopsis thaliana mutants, lacking only one (cyp94b1, cyp94b2, cyp94b3, cyp94c1), two (cyp94b1xcyp94b2, cyp94b1xcyp94b3, cyp94b2xcyp94b3), three (cyp94b1xcyp94b2xcyp94b3) or even four (cyp94b1xcyp94b2xcyp94b3xcyp94c1) CYP94 functionalities. The results obtained in the present study show that CYP94B1, CYP94B2, CYP94B3 and CYP94C1 are responsible for catalyzing the sequential ω-oxidation of JA-Ile in a semi-redundant manner. While CYP94B-enzymes preferentially hydroxylate JA-Ile to 12-hydroxy-JA-Ile, CYP94C1 catalyzes primarily the subsequent oxidation, yielding 12-carboxy-JA-Ile. In addition, data obtained from investigating the triple and quadruple mutants let us hypothesize that a direct oxidation of unconjugated JA to 12-hydroxy-JA is possible in planta. Using a non-targeted metabolite fingerprinting analysis, we identified unconjugated 12-carboxy-JA as novel jasmonate derivative in floral tissues. Using the same approach, we could show that deletion of CYP94-genes might not only affect JA-homeostasis but also other signaling pathways. Deletion of CYP94B1, for example, led to accumulation of metabolites that may be characteristic for plant stress responses like systemic acquired resistance. Evaluation of the in vivo function of the different CYP94-enzymes on the JA-sensitivity demonstrated that particularly CYP94B-enzymes might play an essential role for JA-response, whereas CYP94C1 might only be of minor importance

Biosynthesis of allene oxides in Physcomitrella patens

Background: The moss Physcomitrella patens contains C-18- as well as C-20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results: Our results show that PpAOS1 is highly active with both C-18 and C-20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C-20-substrates, exhibiting trace activity (similar to 1000-fold lower k(cat)/K-M) with C-18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis. Conclusions: Our study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C-18- and C-20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes

Optimized Jasmonic Acid Production by Lasiodiplodia theobromae Reveals Formation of Valuable Plant Secondary Metabolites.

Jasmonic acid is a plant hormone that can be produced by the fungus Lasiodiplodia theobromae via submerged fermentation. From a biotechnological perspective jasmonic acid is a valuable feedstock as its derivatives serve as important ingredients in different cosmetic products and in the future it may be used for pharmaceutical applications. The objective of this work was to improve the production of jasmonic acid by L. theobromae strain 2334. We observed that jasmonic acid formation is dependent on the culture volume. Moreover, cultures grown in medium containing potassium nitrate as nitrogen source produced higher amounts of jasmonic acid than analogous cultures supplemented with ammonium nitrate. When cultivated under optimal conditions for jasmonic acid production, L. theobromae secreted several secondary metabolites known from plants into the medium. Among those we found 3-oxo-2-(pent-2-enyl)-cyclopentane-1-butanoic acid (OPC-4) and hydroxy-jasmonic acid derivatives, respectively, suggesting that fungal jasmonate metabolism may involve similar reaction steps as that of plants. To characterize fungal growth and jasmonic acid-formation, we established a mathematical model describing both processes. This model may form the basis of industrial upscaling attempts. Importantly, it showed that jasmonic acid-formation is not associated to fungal growth. Therefore, this finding suggests that jasmonic acid, despite its enormous amount being produced upon fungal development, serves merely as secondary metabolite

Higher Rate of Tuberculosis in Second Generation Migrants Compared to Native Residents in a Metropolitan Setting in Western Europe

Background: In Western Europe, migrants constitute an important risk group for tuberculosis, but little is known about successive generations of migrants. We aimed to characterize migration among tuberculosis cases in Berlin and to estimate annual rates of tuberculosis in two subsequent migrant generations. We hypothesized that second generation migrants born in Germany are at higher risk of tuberculosis compared to native (non-migrant) residents. Methods: A prospective cross-sectional study was conducted. All tuberculosis cases reported to health authorities in Berlin between 11/2010 and 10/2011 were eligible. Interviews were conducted using a structured questionnaire including demographic data, migration history of patients and their parents, and language use. Tuberculosis rates were estimated using 2011 census data. Results: Of 314 tuberculosis cases reported, 154 (49.0%) participated. Of these, 81 (52.6%) were first-, 14 (9.1%) were second generation migrants, and 59 (38.3%) were native residents. The tuberculosis rate per 100,000 individuals was 28.3 (95CI: 24.0–32.6) in first-, 10.2 (95%CI: 6.1–16.6) in second generation migrants, and 4.6 (95%CI: 3.7–5.6) in native residents. When combining information from the standard notification variables country of birth and citizenship, the sensitivity to detect second generation migration was 28.6%. Conclusions: There is a higher rate of tuberculosis among second generation migrants compared to native residents in Berlin. This may be explained by presumably frequent contact and transmission within migrant populations. Second generation migration is insufficiently captured by the surveillance variables country of birth and citizenship. Surveillance systems in Western Europe should allow for quantifying the tuberculosis burden in this important risk group