On the diverse bonding situations in nanostructures : an ab initio computational study

This computational study investigates diverse bonding situations in nanostructures (carbon nanotubes, fullerenes, metal compounds) spanning a broad range of energies. Weak, dispersive interactions and covalent metal-ligand and metal-metal bonding are examined. The results of efficient density functional calculations are compared to those of correlated wavefunction calculations on model systems. This rigorous validation is crucial in evaluating the balance between computational cost and accuracy

The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor

Characterization of a membrane-bound C-glucosyltransferase responsible for carminic acid biosynthesis in Dactylopius coccus Costa

Carminic acid is a widely applied red colorant that is still harvested from insects because its biosynthesis is not fully understood. Here, the authors identify and characterize a membrane-bound C-glucosyltransferase catalyzing the final step during carminic acid biosynthesis

The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus ‘termite-ball’ and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus-garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defences and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defences such that colony collapse is unavoidable once Escovopsis infections escalate

Molekulare und Chemische Analysen zur Biogenese

0\. Title Page and Table of Contents 1\. General Introduction and Thesis Outline 1 2\. Anthraquinones as Defensive Compounds in Eggs of Galerucini Leaf Beetles: Biosynthesis by the beetle? 17 3\. Presence of Wolbachia in Insect Eggs Containing Antimicrobially Active Anthraquinones 33 4\. Different Polyketide Folding Modes Converge to an Identical Molecular Architecture 49 5\. Defensive Compounds in Insect Eggs: Are Anthraquinones Produced during Egg Development 61 6\. Search for Genes Involved in Anthraquinone Biosynthesis in Galeruca tanaceti 69 7\. Polyketides in Insects 78 8\. Summary 121 9\. Zusammenfassung 125 DanksagungAnthraquinones and anthrones that are not sequestered from food are unusual compounds in insects and only found in leaf beetles of the tribe Galerucini and in scale insects. The major host plants of Galerucini do not contain anthraquinones. Thus, these polyketides might be either produced by the beetle itself or by endosymbiotic microorganisms. The major goal of this thesis was to elucidate the origin of anthrones and anthraquinones in Galerucini leaf beetles with molecular and chemical methods. The tansy leaf beetle, Galeruca tanaceti, was taken as a model Galerucini species containing chrysophanol and chrysazin and the anthrones chrysarobin and dithranol. Endosymbiotic microorganisms were searched in eggs of G. tanaceti. No endosymbiotic bacteria were found except of Wolbachia. This alpha- proteobacterium was absent in the elm leaf beetle (Xanthogaleruca luteola), a closely related Galerucini species containing anthrones/ anthraquinones. However, Wolbachia were present in the anthraquinone-free alder leaf beetle (Agelastica alni). Neither could fungi responsible for anthraquinone production be detected in eggs of G. tanaceti. Furthermore, treatment of adult beetles with antibiotics did not block anthraquinone biosynthesis. This finding supported the hypothesis that no endosymbiotic microorganisms are responsible for anthraquinone production in Galerucini. To elucidate the origin of anthraquinones in G. tanaceti the folding mode of the polyketide chain (octaketide) leading to chrysophanol was measured with NMR techniques. Prokaryotic organisms have another folding mode (type S) than eukaryotic ones (type F). Chrysophanol present in larvae was shown to be synthesised via the eukaryotic folding mode F. Thus, only the beetle itself or a fungus can act as anthraquinone producer. Since no endosymbiotic fungi were detected with molecular techniques, it is considered most likely that the beetle itself is able to produce anthrones and anthraquinones. Knowledge of the anthraquinone production site in G. tanaceti might help to find a prove who (beetle or endosymbiont) produces the anthraquinones. The quantities of some anthrones and anthraquinones decreased significantly during egg development, while others stayed unchanged. These results clearly showed that anthrones and anthraquinones are rather metabolised than produced by the embryo within the eggs. Therefore, the polyketides are transferred by the mother into the eggs and are produced within the female beetle. In a final approach, we searched for genes encoding polyketide synthases (PKS), i.e., enzymes catalysing biosynthesis of polyketides like anthraquinones. No PKS gene responsible for the biogenesis of 1,8-dihydroxylated anthraquinones could be detected so far. Numerous other defensive components and pheromones of insects have been suggested to be polyketides. With the exception of PKS for the polyketide pederin produced by endosymbiotic bacteria, no enzymes responsible for polyketide biosynthesis have been isolated from insects. The precursors of components produced via the polyketide pathway are very similar to those produced via the fatty acid pathway. A brief overview of insect defensive and pheromonal components with (putative) polyketide origin is given. Furthermore, similarities and differences of PKS and fatty acid synthases (FAS) are highlighted.Anthrachinone und Anthrone, die nicht aus der Nahrung sequestriert werden, sind ungewöhnliche Substanzen in Insekten und wurden bisher nur in Blattkäfern der Tribus Galerucini und in Schildläusen gefunden. Die Hauptfutterpflanzen der Galerucini enthalten keine Anthrachinone. Daher werden diese Polyketide entweder vom Käfer selbst oder von endosymbiotischen Mikroorganismen produziert. Das Hauptthema dieser Arbeit beschäftigte sich mit der Untersuchung des Ursprungs der Anthrone und Anthrachinone in Blattkäfern der Tribus Galerucini. Dafür wurden molekulare und chemische Methoden verwendet. Als Modellart eines Blattkäfers der Galerucini wurde der Rainfarnblattkäfer (Galeruca tanaceti) verwendet, der Chrysophanol und Chrysazin und die Anthrone Chrysarobin und Dithranol enthält. In den Eiern von G. tanaceti wurde nach endosymbiotischen Mikroorganismen gesucht. Es konnten keine endosymbiotischen Bakterien außer Wolbachia gefunden werden. Dieses alpha-Proteobakterium konnte im Ulmenblattkäfer (Xanthogaleruca luteola), einer zu den Galerucini nahe verwandten Art, nicht nachgewiesen werden. Im Gegensatz dazu wurden Wolbachien im anthrachinon-freien Erlenblattkäfer (Agelastica alni) gefunden. Außerdem konnten in den Eiern von G. tanaceti keine Pilze, die für eine Anthrachinonproduktion in Frage kämen, nachgewiesen werden. Zusätzlich konnte gezeigt werden, dass eine Behandlung von adulten Käfern mit Antibiotika die Anthrachinonsynthese nicht blockiert. Diese Ergebnisse stützen die Hypothese, dass keine endosymbiotischen Mikroorganismen für die Anthrachinonproduktion in den Galerucini verantwortlich sind. Um den Ursprung der Anthrachinone in G. tanaceti aufzudecken wurde der Faltungstyp der Polyketidkette (Oktaketid), die zum Chrysophanol führt, mit NMR Methoden untersucht. Prokaryoten haben einen anderen Faltungstyp (Typ S) als Eukaryoten (Type F). Es konnte gezeigt werden, dass Chrysophanol aus Larven über den eukaryotischen Faltungstyp F synthetisiert wird. Somit kommt nur der Käfer selbst oder ein Pilz als Anthrachinonproduzent in Frage. Da mit den molekularen Methoden keine Pilze gefunden wurden, ist eine Produktion der Anthrone und Anthrachinone vom Käfer selbst sehr wahrscheinlich. Kenntnisse über den Produktionsort der Anthrachinone in G. tanaceti könnten bei der Suche nach einer Bestätigung, wer diese produziert (Käfer oder Endosymbiont), hilfreich sein. Einige Anthron- und Anthrachinonmengen nahmen signifikant während der Eientwicklung ab, während andere Mengen unverändert blieben. Diese Ergebnisse zeigten, dass die Anthrone und Anthrachinone eher vom Embryo im Ei metabolisiert als von ihm produziert wurden. Die im weiblichen Käfer produzierten Polyketide wurden daher in die Eier transferiert. Abschließend wurde nach Genen für eine Polyketidsynthase (PKS) gesucht. PKS katalysieren die Biosynthese von Polyketiden wie z.B. der Anthrachinone. Es konnten bisher keine PKS Gene gefunden werden, die für eine Biogenese der 1,8-dihydroxylierten Anthrachinone verantwortlich sein könnten. Bei Insekten sind zahlreiche defensive Substanzen und Pheromone bekannt, die möglicherweise ebenfalls Polyketide sind. Bis auf die PKS für das Polyketid Pederin produziert von Endosymbionten wurde noch kein Enzym für die Polyketidsynthese aus Insekten isoliert. Die Bausteine von Substanzen, die über den Polyketidweg produziert werden, sind den Bausteinen von Substanzen sehr ähnlich, die über den Fettsäureweg produziert werden. Zuerst werden die defensiven Substanzen und die Pheromone mit möglichem Polyketidursprung kurz zusammengefasst. Im weiteren werden die Gemeinsamkeiten und Unterschiede von PKS und Fettsäuresynthasen (FAS) beschrieben

The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor

The Structural Basis of Peptide Binding at Class A G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest membrane protein family and a significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influence virtually every aspect of human physiology. About 45% of the members of this family endogenously bind flexible peptides or peptides segments within larger protein ligands. While many of these peptides have been structurally characterized in their solution state, the few studies of peptides in their receptor-bound state suggest that these peptides interact with a shared set of residues and undergo significant conformational changes. For the purpose of understanding binding dynamics and the development of peptidomimetic drug compounds, further studies should investigate the peptide ligands that are complexed to their cognate receptor