Biochemical, biophysical and molecular biological characterization of bacterial poly(cis-1,4-isoprene) oxygenases

Das Thema der Arbeit war die Charakterisierung von RoxA (Rubber oxygenase A) aus Xanthomonas sp. 35Y und Lcp (Latex clearing protein) aus Streptomyces sp. K30. Beide Enzyme initiieren den Abbau von poly(cis-1,4-isopren), indem das Polymer in kleinere Fragmente gespalten und den Produkten dabei Aldehyd- sowie Ketogruppen eingefügt werden. Bei RoxA wurde die Umgebung des N-terminalen Hämzentrums näher untersucht, dem vermuteten aktiven Zentrum, das im „as isolated“ Zustand an der distalen Seite ein O2-Molekül bindet. Anhand der dreidimensionalen Struktur wurden hier verschiedene Aminosäurereste ausgewählt und zielgerichtet mutiert. Tryptophan 291 (W291) liegt auf der proximalen Seite des N-terminalen Hämzentrums und interagiert vermutlich mit dem axialen Histidinliganden (H195). Gereinigtes RoxA-W291Y zeigte eine Aktivität von nur 5% gegenüber dem Wildtyp. Der Aktivitätsverlust und die festgestellten veränderten O2-Bindungseigenschaften lassen sich durch eine verringerte Stabilität oder eine veränderte Positionierung von H195 erklären. Die Aminosäurereste Phenylalanin 301 und 317 (F301 und F317) sind besonders nah und in auffälliger Orientierung zur distalen Seite der N-terminalen Hämgruppe lokalisiert. Eine Substitution der Reste durch andere Aminosäuren führte zu einem Aktivitätsverlust bei den nativ gereinigten Muteinen (von 80% Aktivitätsverlust bei RoxA-F301L bis > 99% bei RoxA-F317Y). Alle Muteine dieser beiden Positionen wiesen veränderte Eigenschaften im UVvis-Spektrum auf, besonders im Bereich der Q-Banden (500 nm - 600 nm), wo im Zustand „as isolated" eine schwächere Absorption als bei RoxA-Wt deutlich wurde. Bei einer Inkubation mit Pyridin, das zu einem Verdrängen des hämgebundenen Sauerstoffs in der Lage ist, zeigte sich bei diesen Muteinen (bis auf RoxA-F301Y) nahezu keine Auswirkung auf das Spektrum. Dies wurde besonders an dem nur schwach ausgeprägten Absorptionssignal bei 549 mm erkennbar, was sie von RoxA-Wt unterscheidet. Die genannten Eigenschaften lassen sich durch eine verminderte O2-Bindungsfähigkeit der Muteine im Vergleich zu RoxA-Wt erklären. In RoxA haben die Aminosäurereste F301 und F317 demnach eine besondere Rolle bei der Bindung und Stabilisierung von molekularem Sauerstoff. Diese Stabilisierung trägt dazu bei, dass RoxA den molekularen Sauerstoff im „as isolated“ Zustand über sehr lange Zeit binden kann, ohne oxidativ inaktiviert zu werden. Diese Besonderheit unterscheidet RoxA von anderen hämhaltigen Dioxygenasen wie der Tryptophan-2,3-Dioxygenase (TDO) und der Indolamin-2,3-Dioxygenase (IDO). Ungewöhnliche Merkmale wurden bei zwei untersuchten Muteinen festgestellt. Bei RoxA-F317Y ist das Sauerstoffatom des Tyrosinatrests axial an das N-terminale Eisenatom gekoppelt, wodurch die Sauerstoffbindestelle blockiert ist. Infolgedessen erklärt sich der vollständige Aktivitätsverlust dieses Muteins. RoxA-F301Y interagiert im Gegensatz zu RoxA-F317Y nicht direkt mit dem Eisenatom der Hämgruppe. Das gebundene O2-Molekül wird hier über Wasserstoffbrücken mit der Hydroxylgruppe des Tyrosins stabilisiert. Aus diesem Grund war ein Verdrängen von hämgebundenem O2 mit Imidazol bei diesem Mutein nicht möglich, was es von RoxA-Wt unterscheidet. Durch die heterologe Expression RoxA-orthologer Proteine aus drei verschiedenen Myxobakterien in Xanthomonas sp. 35Y ΔroxA gelang erstmals eine funktionelle Identifizierung von RoxA in anderen Gram-negativen Bakterien. Gezeigt werden konnte die Aktivität der nativ gereinigten RoxA-orthologen Proteine durch die Klärhofbildung entsprechender rekombinanter Xanthomonas sp. 35Y Stämme auf Latex overlay Agar und einen HPLC-basierten Nachweis des RoxA-Hauptspaltprodukts ODTD. Die UVvis-spektroskopischen Untersuchungen der RoxA-orthologen ergaben vergleichbare Ergebnisse zu RoxA aus Xanthomonas sp. 35Y. Charakteristisch war auch hier die teilweise scheinbare Reduktion nach Inkubation mit Pyridin, die durch das Verdrängen von hämgebundenem Sauerstoff zustande kommt. Im UVvis-Spektrum konnten nach Reduktion mit Dithionit die beiden Hämzentren über die split-α-Bande unterschieden werden, wie es auch bei RoxA aus Xanthomonas sp. 35Y der Fall ist. Ein Unterschied ergab sich bei der Auswirkung von β-Carotin auf die Aktivität, RoxA aus Xanthomonas sp. 35Y wurde hierdurch deutlich weniger inhibiert (55% Aktivität) als RoxA aus Corallococcus coralloides BO35 (10% Aktivität). Dadurch kann eine veränderte Substratspezifität der RoxA-orthologen Proteine im Vergleich zu RoxA aus Xanthomonas sp. 35Y angenommen werden. Die in der hier vorliegenden Arbeit untersuchten Aminosäurereste F301, F317 und W291 sind innerhalb der RoxA-orthologen konserviert und verdeutlichen die Wichtigkeit der Aminosäurereste für diese Enzyme. Das aus dem Gram-positiven Bakterium Streptomyces sp. K30 stammende Lcp konnte in Xanthomonas sp. 35Y ΔroxA funktionell exprimiert werden, was auf Latex overlay Agar durch den Nachweis der Spaltprodukte mit Schiff's-Reagenz sichtbar gemacht wurde. Eine neu etablierte Zwei-Stufen Reinigung von nativem Lcp erlaubte eine erste Charakterisierung des Enzyms. Das pH-Optimum lag zwischen 7 und 8 in 100 mM Kaliumphosphatpuffer. Detergenzien inhibierten die Aktivität vollständig, Imidazol und Dithiothreitol teilweise. Bis auf Diethyldithiocarbamat hatten andere Chelatbildner keinen inhibitorischen Effekt auf die Aktivität von Lcp. Bei 37°C zeigte sich eine deutlich höhere temperaturabhängige Inaktivierung für Lcp als für RoxA. Die Ausbeute war mit 1,5 mg Lcp pro 12 l Kultur sehr gering, für weitere Untersuchungen wurde daher Lcp N-terminal mit einem Strep-Tag ausgestattet und in E. coli heterolog exprimiert. Die Ausbeute steigerte sich so auf das 40-fache (5 mg/l). Die spezifische Aktivität von Lcp wurde auf Basis des verbrauchten Sauerstoffs im Reaktionsansatz ermittelt (23°C: 1,5 U/mg; 37°C: 4,6 U/mg). Über einen spektroskopischen Test und auch mittels MALDI-TOF Analyse (Matrix-assisted laser desorption/ionization - time of flight) ließ sich Lcp den b-Typ Cytochromen zuordnen. Spektroskopische Untersuchungen mit Imidazol und Kohlenstoffmonoxid offenbarten eine oxidierte und damit sauerstofffreie Hämgruppe (Fe3+), was im Gegensatz zur Hämgruppe des aktiven Zentrums von RoxA steht (Fe2+--O2).The subject of this thesis was the characterization of RoxA (rubber oxygenase A) from Xanthomonas sp. 35Y and Lcp (latex clearing protein) from Streptomyces sp. K30. Both enzymes initiate the degradation of poly(cis-1,4-isoprene) by cleaving the polymer to aldehyde- and keto-group containing fragments. In RoxA the surrounding of the N-terminal heme center was studied. This is the supposed active site of the enzyme, it binds a dioxygen molecule in the „as isolated“ state. The three-dimensional structure of RoxA was used to select several amino acid positions for site-directed mutagenesis. Tryptophan 291 (W291) is located at the proximal site of the N-terminal heme center. It presumably interacts with the axial histidine ligand (H195). Purified RoxA-W291Y showed an activity of 5% compared to the wildtype. The decreased activity as well as the altered oxygen binding properties seem to be caused by modified positioning of H195 or stability loss of the enzyme. The amino acid residues phenylalanine 301 and 317 (F301, F317) are located in close distance to the distal site of the N-terminal heme center. Substitution with other amino acids resulted in decreased activity of the purified muteins (from 80% activity loss for RoxA-F301L to >99% for RoxA-F317Y). All muteins showed altered properties in the UVvis-spectrum, particularly in the region of the Q-bands (500 nm - 600 nm). Here, a lower absorption as in RoxA-Wt was observed. In contrast to RoxA-Wt, an incubation with pyridine, which can replace the heme-bound dioxygen, did not result in altered UVvis spectra of the muteins (except RoxA-F301Y). The reason for the stated properties of the muteins can be found in a decreased O2-binding ability compared to RoxA-Wt. Thus, the amino acid residues F301 and F317 are important for binding and stabilizing the heme-bound dioxygen. This contributes to the high stability of dioyxgen-bound RoxA, a property that distinguishes this enzyme from other heme containing dioxygenases such as tryptophan-2,3-dioxygenase (TDO) or indolamine-2,3-dioxygenase (IDO). Unusual characteristics were found for two muteins. In RoxA-F317Y, the oxygen atom of the tyrosinate residue interacts with the N-terminal iron, thereby blocking the dioxygen binding pocket. This explains the complete loss of enzymatic activity. In contrast to RoxA-F317Y, the tyrosine residue of RoxA-F301Y does not interact with the iron atom of the heme group. Here, the bound dioxygen is stabilized by hydrogen bonds with the hydroxyl-group of tyrosine. The heterologous expression of RoxA-orthologous proteins in Xanthomonas sp. 35Y, obtained from three different myxobacteria, allowed a functional identification of RoxA in other Gram-negative bacteria. The activity of these proteins was demonstrated with the appearance of clearing zones around the corresponding recombinant Xanthomonas sp. 35Y strains and by HPLC-based detection of the cleavage product ODTD. The UVvis-spectroscopic properties of the RoxA-orthologous proteins were similar to RoxA from Xanthomonas sp. 35Y. Incubation with pyridine resulted in a partial reduction in the spectrum, triggered by replacement of the N-terminal bound dioxygen with this compound. Chemical reduction with dithionite resulted in a split-α-band, similar to RoxA from Xanthomonas sp. 35Y. A difference between RoxA and the orthologous proteins was shown after incubation with the inhibitor β-carotine (55% activity for RoxA from Xanthomonas sp. 35Y, 10% activity for RoxA from Corallococcus coralloides BO35). This suggested a different substrate specificity of the RoxA-orthologous proteins compared to RoxA from Xanthomonas sp. 35Y. The amino acid residues F301, F317 and W291, that were studied in this work, are conserved among the different RoxA proteins. This demonstrates the significance of these residues for RoxAs. Lcp of the Gram-positive bacterium Streptomyces sp. K30 was functionally expressed in Xanthomonas sp. 35Y ΔroxA. The cleavage products were detected with Schiff's reagent on latex overlay agar. A new developed two-step purification of native Lcp was established, this allowed a first characterization of this enzyme. The pH-optimum was determined between 7 and 8 in 100 mM potassium phosphate buffer. Detergents completely inhibited the activity of Lcp, imidazole and dithiothreitol partially. With the exception of diethyldithiocarbamate, chelators had no inhibitory effect on Lcp activity. An incubation at 37°C showed that Lcp is stronger affected by heat dependent inactivation than RoxA. The Lcp yield was increased 40-fold with a Strep-Tag construct and expression in E. coli (5 mg/l). The specific activity for Lcp was determined (23°C: 1,5 U/mg; 37°C: 4,6 U/mg). Lcp was assigned as b-type cytochrome by spectroscopic and MALDI-TOF analysis. Moreover, incubation of Lcp with imidazole or carbon monoxide revealed an oxidized, oxygen-free heme-group (Fe3+). This is in sharp contrast to the active site heme of RoxA (Fe2+--O2)

Sustainable reserve power from demand response and fluctuating production - two Danish demonstrations

The Danish grid is moving from a system based on centralized fossil fueled power plants to a system based on renewable energy where wind is a ma-jor energy source. This raises a number of challenges. A main challenge is that the centralized power plants currently are the main providers of re-serve power. Alternative sources of flexibility are consequently needed as the conventional power plants are being replaced with fluctuating renewable energy sources. In this paper we present results from two key demonstra-tions which illustrate that alternative sources of flexibility exist and that this flexibility can be utilized for reserve power. In the first demonstration, a portfolio of inhabited households heated with heat pumps are remotely mon-itored and controlled such that the aggregate consumption follows a power reference. This experiment is conducted over a full week where an hourly power reference is tracked while the comfort of the inhabitants is ensured. In the second demonstration, an operational wind power plant is regulated to provide a system-stabilizing response. This experiment is conduced over a two-hour period where the wind power plant follows a 5-minute power ref-erence. Together, the two demonstrations illustrate that both consumption and fluctuating production can contribute as sources of reserve power and a sustainable alternative to conventional fossil fueled power plants in the future grid

Correction: Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium Steroidobacter cummioxidans sp. nov., strain 35Y.

[This corrects the article DOI: 10.1371/journal.pone.0197448.]

Metabolic and taxonomic insights into the Gram-negative natural rubber degrading bacterium <i>Steroidobacter cummioxidans</i> sp. nov., strain 35Y

<div><p>The pathway of rubber (poly [<i>cis</i>-1,4-isoprene]) catabolism is well documented for Gram-positive rubber degraders but only little information exists for Gram-negative species. The first documented potent rubber degrading Gram-negative strain is <i>Xanthomonas</i> sp. strain 35Y that uses extracellular rubber oxygenases for the initial cleavage of the polyisoprene molecule. However, neither the exact phylogenetic position of <i>Xanthomonas</i> sp. strain 35Y nor the catabolic pathway of the primary polyisoprene cleavage products have been investigated. In this contribution, we started to address both these issues by a comprehensive taxonomic characterization and by the analysis of the draft genome sequence of strain 35Y. Evaluation of the 16S rRNA gene sequence pointed to a borderline taxonomic position of strain 35Y as a novel species of the genus <i>Steroidobacter</i>. Further, substantial differences in the genotypic properties of strain 35Y and the members of the genus <i>Steroidobacter</i>, including average nucleotide identity (ANI) and <i>in silico</i> DNA-DNA hybridization (DDH), resolved the taxonomic position of strain 35Y and suggested its positioning as a novel species of the genus <i>Steroidobacter</i>. This was further confirmed by comparative analysis of physiological and biochemical features of strain 35Y with other members of the genus <i>Steroidobacter</i>. Thus, we conclude that strain 35Y represents a novel species of the genus <i>Steroidobacter</i>, for which we propose the designation <i>Steroidobacter cummioxidans</i> sp. nov., strain 35Y^T. A comprehensive analysis of the draft genome of <i>S</i>. <i>cummioxidans</i> strain 35Y revealed similarities but also substantial differences to rubber degrading Gram-positive counterparts. In particular, the putative transporters for the uptake of polyisoprene cleavage products differ from Gram-positive rubber degrading species. The draft genome sequence of <i>S</i>. <i>cummioxidans</i> strain 35Y will be useful for researchers to experimentally verify the predicted similarities and differences in the pathways of polyisoprene catabolism in Gram-positive and Gram-negative rubber degrading species.</p></div

Carrier's Liability in International Carriage of Goods

The purpose of this thesis was to analyse and compare isseu of carrier's liability in international carriage of goods. Thesis is devided in seven parts, which offers outline of carrier's liability in particular modes of carriage. First part deals with term contract for the carriage according civil code, parts of this contract as well as another contracts which aim is carriage. Furthermore deals with term liability and put outline of distinction among strict liability and liability for fault. Last subchapter of first part describes term carriage. Second part describes legal framework of contract for the international carriage of goods and specifies distinction among choice of law and direct method. Remaining parts refer to carrier's liability according international treaties concerned with particular modes of carriage, i. e. international carriage of goods by road, by rail, by air, by sea and by inland waterways. Bigger attention is dedicated to third part which deals with carrier's liability in international carriage of goods by road. Emphasis to this part is given, because international carriage of goods by road is the most frequently one - especially from Czech point of view. International carriage of goods by road is for sixty years ruled by CMR Convention which was amended only twice. This mode..

Characteristics of <i>Steroidobacter cummioxidans</i> strain 35Y (35Y), <i>Steroidobacter flavus</i> CPCC 100154 (CPCC 100154), <i>Steroidobacter agariperforans</i> KA5-B (KA5-B), <i>Steroidobacter denitrificansis</i> DSM 18526 (DSM 18526).

<p>Characteristics of <i>Steroidobacter cummioxidans</i> strain 35Y (35Y), <i>Steroidobacter flavus</i> CPCC 100154 (CPCC 100154), <i>Steroidobacter agariperforans</i> KA5-B (KA5-B), <i>Steroidobacter denitrificansis</i> DSM 18526 (DSM 18526).</p

Predicted natural rubber degradation pathway of <i>Steroidobacter cummioxidans</i> strain 35Y.

<p>The pathway comprises of five steps: (1) biosynthesis of rubber oxygenases and extracellular oxidative cleavage of polyisoprene chains, (2) import of oligoisoprenes, (3) β-oxidation, (4) acetyl-CoA and propionyl-CoA metabolism, and (5) anaplerotic reactions and gluconeogenesis. RoxA: Rubber oxygenase A, RoxB: Rubber oxygenase B, ODTD: 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al, Mce: Mammalian cell entry protein, Mla: An ABC transport system that maintains lipid asymmetry, TBDRs: TonB-dependent outer membrane receptors, FACS: fatty acyl coenzyme A (CoA) synthetase: FadL: It encodes outer membrane proteins/ transporters involved in long-chain fatty acid, TCA: Tricarboxylic acid cycle, PEP: Phosphoenolpyruvate.</p

Phylogenetic relationships between <i>Steroidobacter cummioxidans</i> sp. nov., strain 35Y and members of the family <i>Sinobacteraceae</i> on the basis of 16S rRNA gene sequences.

<p>The phylogenetic tree was constructed by using the maximum likelihood method, and the 16S rRNA gene sequence of <i>Nitrosomonas aestuarii</i> Nm69 was used as the outgroup. Numbers at nodes indicate levels of bootstrap support (%) based on a maximum likelihood analysis of 1000 resampled datasets; values of less than 50 % are not shown. Bar, 0.02 nucleotide substitutions per site. Solid lines represent the lengths of branches; dotted lines are used to align the tip labels for better visualization; circle represents the node.</p

Basic genome features of <i>Steroidobacter cummioxidans</i> strain 35Y.

<p>Basic genome features of <i>Steroidobacter cummioxidans</i> strain 35Y.</p