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Protein biosynthesis is a conserved process, essential for life. Proteins are assembled from single amino acids according to their genetic blueprint in the form of a messenger ribonucleic acid (mRNA). Peptide bond formation is catalyzed by ancient ribonucleic acid (RNA) residues within the supramolecular ribosomal complex, which is organized in two dynamic subunits (Ramakrishnan, 2014). Each subunit comprises large ribosomal RNA (rRNA) molecules and several dozens of peripheral proteins. mRNA translation has been divided into three phases, namely translation initiation, elongation and termination in biochemistry textbooks. During initiation, the ribosomal subunits assemble into a functional ribosome on an activated mRNA and acquire the first transfer RNA (tRNA), an adapter between the start codon on the mRNA and the N-terminal methionine of the protein (Hinnebusch and Lorsch, 2012). During elongation, the ribosome translocates along the mRNA exposing one codon after the other, and amino acids are delivered to the ribosome by the respective tRNAs, and attached to the nascent polypeptide chain. During termination, the polypeptide is released and the ribosome remains loaded with mRNA and tRNA at the end of the open reading frame for the translated gene (Hellen, 2018). Bacterial ribosomes are subsequently recycled by a specific ribosome recycling factor and the small ribosomal subunit is simultaneously consigned to initiation factors for a next round of translation – rendering bacterial translation as a cyclic process with an additional ribosome recycling phase. However, the process of ribosome recycling remained enigmatic in Eukarya and Archaea until the simultaneous discovery of the twin-ATPase ABCE1 as the major ribosome recycling factor. Strikingly, ABCE1 has initially been shown to participate in translation initiation (Nürenberg and Tampé, 2013). Thus, closing the translation cycle by revealing the detailed molecular mechanism of ABCE1 and its role for translation initiation are the two goals of this research.
Beyond the plenitude of well-studied translational GTPases, ABCE1 is the only essential factor energized by ATP, delivering the energy for ribosome splitting via two nucleotide-binding sites. Here, I define how allosterically coupled ATP binding and hydrolysis events in ABCE1 empower ribosome recycling. ATP occlusion in the low-turnover control site II promotes formation of the pre-splitting complex and facilitates ATP engagement in the high-turnover site I, which in turn drives the structural re- organization required for ribosome splitting. ATP hydrolysis and ensuing release of ABCE1 from the small subunit terminate the post-splitting complex. Thus, ABCE1 runs through an allosterically coupled cycle of closure and opening at both sites consistent with a processive clamp model. This study delineates the inner mechanics of ABCE1 and reveals why various ABCE1 mutants lead to defects in cell homeostasis, growth, and differentiation (Nürenberg-Goloub et al., 2018).
Additionally, a high-resolution cryo-electron microscopy (EM) structure of the archaeal post-splitting complex was obtained, revealing a central macromolecular assembly at the crossover of ribosome recycling and translation initiation. Conserved interactions between ABCE1 and the small ribosomal subunit resemble the eukaryotic complex (Heuer et al., 2017). The conformational state of ABCE1 at the post-splitting complex confirms the molecular mechanism of ribosome recycling uncovered in this study. Moving further along the reaction coordinate of cellular translation, I reconstitute the complete archaeal translation initiation pathway and show that essential archaeal initiation factors are recruited to the post-splitting complex by biochemical methods and cryo-EM structures at intermediate resolution. Thus, the archaeal translation cycle is closed, following its bacterial model and paving the way for a deeper understanding of protein biosynthesis.
Die Paarverteilungsfunktion (PDF) beschreibt die Wahrscheinlichkeit, zwei Atome eines Materials in einem Abstand r voneinander zu finden. Diese Methode bewährt sich seit längerer Zeit zur Untersuchung von Gläsern, Flüssigkeiten, amorphen, stark fehlgeordneten und nanokristallinen anorganischen Substanzen. Die Anwendung für organische Substanzen ist jedoch relativ neu, mit etwa 20 Veröffentlichungen und Patenten insgesamt.
Im Rahmen dieser Dissertation wurden zwei Methoden zur Strukturverfeinerung und Strukturlösung organischer Substanzen anhand von PDF-Daten erfolgreich entwickelt und an diversen Beispielen validiert. Als erster Schritt hierzu wurde eine Methodenverbesserung vorgenommen. Hierbei handelte es sich um eine Verbesserung der Simulation der PDF-Kurven organischer Verbindungen anhand eines gegebenen Strukturmodells. Mit Hilfe der bisherigen Methoden können die PDF-Kurven anorganischer Substanzen erfolgreich simuliert werden. Für organische Substanzen werden bei Anwendung der bisherigen Methode die Signalbreiten der intramolekularen und intermolekularen Beiträge zu der PDF-Kurve falsch wiedergegeben, dies führt zu einer schlechten Anpassung der simulierten PDF-Daten and die experimentellen PDF-Daten. Deshalb wurde ein neuer Ansatz entwickelt, in welchem für die Berechnung der intramolekularen Beiträge zum PDF-Signal ein anderer isotroper Auslenkungsparameter verwendet wurde, als bei der Berechnung der intermolekularen Beiträge zum PDF-Signal. Mit diesem Ansatz konnte eine sehr gute Simulation der PDF-Kurve für alle Testbeispiele erzielt werden. Zur Strukturverfeinerung organischer Substanzen anhand von PDF-Daten wurden zwei Ansätze entwickelt: der Rigid-Body-Ansatz zur Behandlung starrer organischer Moleküle und der Restraint-Ansatz zur Behandlung flexibler organischer Moleküle.
Neben methodischen Entwicklungen wurden in dieser Arbeit zwei weitere Untersuchungen organischer Verbindungen mittels PDF-Analyse durchgeführt.
Es wurden drei, auf unterschiedliche Weise hergestellte, amorphe Proben des Wirkstoffes Telmisartan untersucht. Des Weiteren wurde mittels PDF-Analyse eine pharmazeutische Nanosuspension untersucht.
Die chemischen und physikalischen Eigenschaften eines Festkörpers sind vom inneren Aufbau des Festkörpers abhängig. Die Methode der Wahl zur Bestimmung von Kristallstrukturen sind Beugungsexperimente. Fehlordnungen in den Kristallstrukturen werden mit Beugungsexperimenten häufig nur unzureichend ausgewertet oder ignoriert. In dieser Arbeit wurden die (möglichen) Stapelfehlordnungen der Aminosäuren DL-Norleucin und DL-Methionin, sowie von Chloro (phthalocyaninato)aluminium(III) untersucht. Dazu wurden Gitterenergieminimierungen mit Kraftfeld- und quantenchemischen Methoden an einem Satz geordneter Modellstrukturen durchgeführt.
In den Kristallstrukturen der α- und β-Phasen von DL-Norleucin ordnen sich die Moleküle in Doppelschichten an und bilden jeweils eine Schichtstruktur mit unterschiedlicher Stapelsequenz. Röntgenbeugungsexperimente an Kristallen dieser Verbindung zeigen charakteristische diffuse Streuung. Die durchgeführten Gitterenergieminimierungen reproduzieren die experimentelle Stabilitätenreihenfolge der beiden Polymorphe von DL-Norleucin. Die berechneten Gitterenergien zeigen, dass es für DL-Norleucin bevorzugte Stapelsequenzen gibt. Die Gitterenergien und Molekülstrukturen einer einzelnen Doppelschicht sind dabei von der Anordnung benachbarter Doppelschichten abhängig. Zudem wurden Strukturmodelle mit Stapelsequenzen aufgebaut, die aus kristallographischer Sicht möglich sind, jedoch experimentell nicht beobachtet wurden, und deren Gitterenergie berechnet. Diese Stapelsequenzen liefern im Vergleich zu den energetisch günstigsten Stapelsequenzen einen signifikanten Energieverlust und treten daher selten auf. Ausgehend von den Ergebnissen der Gitterenergieminimierungen mit DFT-D-Methoden wurden Stapelwahrscheinlichkeiten mit Hilfe der Boltzmann-Statistik berechnet. Es wurde ein großes geordnetes Modell mit einer Stapelsequenz gemäß der Stapelwahrscheinlichkeiten aufgebaut. Dieses Modell wurde verwendet, um Beugungsexperimente zu simulieren und mit experimentellen Daten zu vergleichen. Die theoretischen und experimentellen Beugungsdaten waren in guter Übereinstimmung.
Die Moleküle in den Kristallstrukturen der α- und β-Phasen von DL-Methionin bilden Doppelschichten. Die beiden Phasen unterscheiden sich in der Stapelung der Doppelschichten und der Molekülkonformation. Es wurden Gitterenergieminimierungen sowohl mit Kraftfeld-Methoden als auch mit DFT-DMethoden an geordneten Modellen mit unterschiedlichen Stapelsequenzendurchgeführt. Die experimentell bestimmte Stabilitätenreihenfolge der Polymorphe von DL-Methionin bei tiefen Temperaturen wurde durch die Ergebnisse der kraftfeldbasierten Rechnungen reproduziert. Die Modellstrukturen wurden während den Rechnungen moderat verzerrt. Die Bandbreite der relativen Energien aller Modelle ist relativ gering, sodass eine Stapelfehlordnung aus thermodynamischer Sicht nicht ausgeschlossen werden kann. In der Regel liefern Gitterenergieminimierungen mit DFT-D Methoden genauere Ergebnisse. Die Modellstrukturen wurden während den Rechnungen nur leicht verzerrt. Allerdings unterscheidet sich das Energieranking zwischen den Kraftfeld- und DFT-D-Methoden deutlich. Die experimentell bestimmte Stabilitätenreihenfolge der Polymorphe von DL-Methionin wurde mit DFT-D-Methoden nicht reproduziert. Die Energieunterschiede zwischen den beiden Polymorphen (ΔE = 1,60 kJ∙mol−1 (DFT-D2) bzw. ΔE = 0,83 kJ∙mol−1 (DFT-D3)) sind relativ gering und liegen im Fehlerbereich der Methode. Die Bandbreite der relativen Energien aller Strukturmodelle beträgt nur etwa 1,8 kJ∙mol−1. Auf dieser Grundlage ist eine Stapelfehlordnung in den Kristallstrukturen von DL-Methionin möglich, jedoch nicht experimentell beobachtet. Nicht nur die Kraftfeld-,sondern auch die DFT-D-Methoden scheinen für die Berechnung der Gitterenergien für das System DL-Methionin nicht genügend genau zu sein. Die erhaltenen relativen Energien sollten daher mit Vorsicht betrachtet werden.
Chloro(phthalocyaninato)aluminium(III) (AlPcCl) bildet eine Schichtstruktur, in der sich die Moleküle zu Doppelschichten zusammenlagern. Die 1984 durchgeführte Kristallstrukturbestimmung [98] lieferte auf Grund der schlechten Datenqualität nur eine ungenaue Kristallstruktur. Die asymmetrische Einheit enthält zwei Moleküle, von denen das eine Molekül geordnet, das andere fehlgeordnet ist. Die Kristallstruktur von AlPcCl ist fehlgeordnet, weil eine einzelne Doppelschicht von Molekülen eine tetragonale P4/n-Symmetrie aufweist, die vier symmetrieäquivalente Möglichkeiten bietet, eine zweite Doppelschicht auf einer ersten Doppelschicht zu platzieren. Mit Hilfe der OD-Theorie wurde ein Satz geordneter Modelle mit verschiedenen Stapelsequenzen aufgestellt und die Gitterenergie zunächst mit Kraftfeld-Methoden und anschließend mit DFT-DMethoden berechnet. Auf Grund unzureichender Parametrisierung, musste das Kraftfeld an das System AlPcCl angepasst werden. Die Modellstrukturen werden während den Kraftfeld-Rechnungen nur leicht verzerrt. Die berechneten Gitterenergien hängen allerdings stark von der verwendeten Parametrisierung und den Atomladungen ab und sollten daher mit Vorsicht betrachtet werden. Genauere Ergebnisse erzielten Gitterenergieminimierungen mit DFT-D-Methoden. Die verschiedenen Stapelsequenzen haben eine ähnliche Energie, was die Stapelfehlordnung in der Kristallstruktur von AlPcCl erklärt. Die Überlagerung der vier energetisch günstigsten geordneten Stapelsequenzen führt zu einer gemittelten Struktur, die sehr gut die fehlgeordnete experimentelle Kristallstruktur von AlPcCl erklärt.
Ziel dieser Doktorarbeit war es, die Bedeutung der Kristallstrukturbestimmung aus Pulverdaten (SDPD) herauszuarbeiten und etwaige Grenzen durch neue Methodenentwicklungen zu erweitern, insbesondere bei Analyse der Paarverteilungsfunktion (PDF).
Die Effizienz der SDPD konnte anhand der erfolgreich gelösten Kristallstruktur von Carmustin (1,3 Bis-2-chlorethyl-1-nitrosoharnstoff, C5H9Cl2N3O2) aufgezeigt werden. [CS01]
Die Grenzen der SDPD wurden ausgelotet und erfolgreich erweitert. Nach weit verbreiteter kristallographischer Meinung ist die Strukturlösung mittels des simulierten Temperns (simulated annealing, SA) bei mehr als 25 zu bestimmenden Parametern problematisch oder unmöglich. Die pharmazeutischen Salze Lamivudin-Camphersulfonat (LC) und Aminogluthethimid-Camphersulfonat (AC) konnten, trotz ihrer hohen Anzahl an Freiheitsgraden von 31 für LC bzw. 37 für AC erfolgreich bestimmt werden. Die Strukturlösung von AC war herausfordernd und nicht direkt bei Anwendung der SA-Methode möglich. Nach einer intensiven Fehleranalyse stellte sich heraus, dass nicht die Grenzen der SA-Methode ausschlaggebend für das anfängliche Scheitern der Strukturlösung waren, sondern falsch extrahierte Intensitäten des vorangegangenen Pawley-Fits. Nach Behebung dieser Fehlerquelle war die Strukturlösung von AC problemlos. [CS02]
Mittels SDPD kann die absolute Konfiguration chiraler Verbindungen nicht direkt bestimmt werden. Durch Kristallisation der zu bestimmenden chiralen Verbindung mit einem chiralen Gegenion bekannter Konformation in einer simplen Säure-Base-Reaktion zu einem diastereomeren Salz und nachfolgender SDPD konnte eine neue Methode entwickelt werden, um die Konfigurationsbestimmung aus Pulverdaten zu ermöglichen. Diese Methode wurde anhand der drei pharmazeutischen Salze (R)-Flurbiprofen-(R)-Chinin (FQ), (2R5S)-Lamivudin-(R)-Camphersulfonat (LC) und (R)-Aminogluthethimid-(R)-Camphersulfonat (AC) aufgezeigt: In allen drei Fällen konnte die korrekte Konfiguration des pharmazeutischen Wirkstoffes mit den hierfür entwickelten Kriterien erfolgreich bestimmt werden. [CS03, CS04]
Durch Kombination der klassischen SDPD mit neuen methodischen Ansätzen konnten die Kristallstrukturen der schlecht kristallinen organischen Pigmente 2-Monomethylchinacridon (MMC, C21H14N2O2) und 4,11-Difluorchinacridon (DFC, C20H10N2O2F2) bestimmt werden, obwohl aufgrund ihrer geringen Kristallqualität keine sinnvolle Indizierung möglich war.
Für die Kristallstrukturbestimmung von DFC lieferte der neu entwickelte Global-Fit des Programms FIDEL mögliche Strukturmodelle mit ähnlich guter Übereinstimmung an das experimentelle Pulverdiagramm. Die Rietveld-Verfeinerung der Strukturmodelle in Kombination mit der Anpassung der Kristallstruktur an die PDF-Daten und kraftfeldbasierter Gitterenergieminimierung konnte einen geeigneten Strukturrepräsentanten von DFC liefern. [CS05, CS06]
Im Fall von MMC war eine Kombination der Methoden von Rietveld-Verfeinerung, Verfeinerung an die PDF-Daten und Gitterenergieminimierung zielführend zur Bestimmung der Orientierungs-Fehlordnung von MMC im Kristall. MMC ist hierbei die erste organische Verbindung, deren Fehlordnung durch Anpassung an die PDF bestimmt werden konnte. [CS07]
Große Erfolge konnten bei der Methodenentwicklung der PDF-Analyse erzielt werden. Die Bestimmung von Kristallstruktur organischer Verbindungen durch Anpassung an die PDF ohne vorherige Kenntnis der Gitterparameter oder Raumgruppe wurde durch die Entwicklung des PDF-Global-Fits erreicht. Lediglich die PDF-Kurve und eine Molekülstruktur werden als Input benötigt. Die Strukturlösung beruht auf einem globalen Optimierungs-Ansatz, bei welchem in ausgewählten Raumgruppen Zufallsstrukturen erzeugt werden. Die Zufallsstrukturen werden mit den experi¬mentellen Daten verglichen und entsprechend ihres Ähnlichkeitsindexes, basierend auf der Kreuz-Korrelation, sortiert. [CS08, CS09] Die vielversprechendsten Kandidaten werden in einem einge¬schränkten simulierten annealing-Ansatz an die experimentelle PDF angepasst. Eine nachfolgende Strukturverfeinerung der besten Strukturmodelle liefert die korrekte Kristallstruktur. Der Erfolg des PDF-Global-Fits wurde am Beispiel der Barbitursäure aufgezeigt: Ausgehend von 300 000 Zufallsstrukturen konnte die korrekte Kristallstruktur von Barbitursäure bestimmt werden. Barbitursäure ist hierdurch die erste organische Verbindung, deren Lokalstruktur durch Anpassung an die PDF bestimmt wurde, ohne Input oder Vorgabe von Gitterparametern oder Raumgruppe.[CS10]
In this thesis, we characterized megasynthases such as fatty acid synthases (FASs) and polyketide synthases. The obtained insights into structure and function were used to engineer such systems to produce new-to-nature compounds.
The in vitro characterization of megasynthases requires reproducible access to these enzymes in high quality. Therefore, we established purification strategies for the yeast FAS and the methylsalicylic acid synthase (MSAS) from Saccharopolyspora erythraea (SerMSAS) and applied the latter one on MSAS from Penicillium patulum (PenPaMSAS) and on 6-deoxyerythronolide B synthase (DEBS) module 6. With the purified samples, we were able to obtain initial structural data for SerMSAS and solve the complete structure of the yeast FAS (PDB: 6TA1). On the example of the yeast FAS, we could show that the sample can suffer from adsorption to the water-air interface during the grid preparation for electron microscopy and presented how the use of graphene-based grids can overcome this problem. The combined structural and functional analysis of the yeast FAS showed that the structural domains trimerization module and dimerization module 2 are not essential for the assembly of the whole system. Therefore, they can potentially be used for domain exchange approaches. The in-depth functional analysis of SerMSAS revealed that not SerMSAS itself releases the product, but a 3-oxoacyl-(acyl-carrier protein) synthase like enzyme within the gene cluster transfers 6-methyl salicylic acid from SerMSAS to another carrier protein for subsequent modifications. In contrast, we showed that PenPaMSAS can release its product by hydrolysis and that non-native substrates can be incorporated although at significantly slower turnover rates compared to the native starter substrate. Our further investigation demonstrated that the substrate specificity of the acyltransferase (AT) is a critical factor for the incorporation of non-native substrates.
With the insight from the functional and structural characterization, we engineered megasynthases for the biosynthesis of natural product derivatives. We targeted the AT of PenPaMSAS for active site mutagenesis and discovered a mutant which can transfer non-native substrates significantly faster (~200-300%). Additionally, the malonyl/acetyl transferase (MAT) of the mammalian FAS was used as a promising target for protein engineering because of its previously reported properties including polyspecificity, fast transfer kinetics, robustness, and plasticity. We showed that the MAT can transfer fluorinated substrates and accept the acyl carrier protein of DEBS module 6. By exchanging the substrate specific AT of DEBS with the polyspecific MAT of the mammalian FAS, we demonstrated an efficient DEBS/FAS hybrid and an optimal truncation site for the applied ATs. In contrast to the wild type system, the DEBS/FAS enzyme was able to synthesize demethylated and fluorinated derivatives. The production and purification of a fluoro-methyl-disubstituted polyketide was of particular interest, as it has a high potential for the generation of new drugs and shows the potential of protein engineering. Furthermore, the incorporation of the disubstituted substrate had important implication in the mechanistic details of the ketosynthase-mediated C-C bond formation.
Polyketide synthases (PKSs) are large megaenzymes that occur in bacteria, fungi, and plants and produce polyketides, a class of secondary metabolites. Many polyketide natural products exhibit high biological activities e.g. as antibiotics or anti-fungal compounds. The modular architecture of assembly line PKSs makes them exciting targets for engineering approaches via the exchange of whole modules or single domains. Although many engineering attempts have been pursued over the last three decades, the resulting chimeric PKSs often exhibit decreased turnover rates or diminished product yields.
In this thesis, new approaches to engineer chimeric PKSs were explored, each targeting a different aspect of the chimeric system: First the relative contribution of protein-protein and protein-substrate recognition on the turnover of chimeric PKS was assessed, revealing the importance of protein-protein interactions between the acyl carrier protein (ACP) and the ketosynthase (KS) domain in the chain translocation step. Directed evolution experiments followed to optimize the protein-protein interaction across a chimeric interface. Additionally, different junction sites for the generation of chimeric PKSs were compared, showing the ability for recombination without interfering with the chain translocation reaction, and highlighting the use of SYNZIP domains to bridge PKS modules. To optimize chimeric PKSs even further, multipoint mutagenesis of KS domains was established, with positive effects on the activity of chimeric systems.
To support engineering attempts, several structure elucidation techniques were combined with in silico modeling to characterize the architecture of a PKS module and the domain-domain interactions within it. Preliminary results show a strong conformational flexibility of the PKS module and the great potential of these techniques to define the multitude of transient interactions in PKS modules.
Bacteria are highly organized organisms which are able to adapt to and propagate under a multitude of environmental conditions. Propagation hereby requires reliable chromosome replication and segregation which has to occur cooperatively with other cellular processes such as transcription, translation or signaling. Several mechanisms were proposed for segregation of the Escherichia coli (E. coli) chromosome, for example a mitotic-like active segregation model or entropy-based passive chromosome segregation. Another segregation model suggests coupled transcription, translation and insertion of membrane proteins (termed "transertion"), which links the replicating chromosome (nucleoid) to the growing cell cylinder.
Fluorescence microscopy was widely used to provide evidence for a distinct segregation model. However, the dynamic nature of bacterial chromosomes, the small bacterial size and the optical resolution limit of ~ 200-300 nm impair unveiling the underlying mechanisms. With the emergence of super-resolution fluorescence microscopy techniques and advanced labeling methods, a new toolbox became available enabling scientists to visualize biomolecules and cellular processes in unprecedented detail. Single-molecule localization microscopy (SMLM) represents a set of super-resolution microscopy techniques which relies on the temporal separation of the fluorescence signal and detection of single fluorophores. Separation can be achieved using photoactivatable or -convertible fluorescent proteins (FPs) in photoactivated localization microscopy (PALM), photoswitchable organic dyes in direct stochastic optical reconstruction microscopy (dSTORM) or dynamically binding fluorescent probes in point accumulation for imaging in nanoscale topography (PAINT). In all these techniques, the fluorescence emission pattern of single fluorophores is spatially localized with nanometer-precision. An artificial image is finally reconstructed from the coordinates of all single fluorophores detected. This provides a spatial resolution of ~ 20 nm, which is perfectly suited to investigate cellular processes in bacteria. In this thesis, different SMLM techniques were applied to study fundamental processes in E. coli. This includes determination of protein copy numbers and distributions as well as the nanoscale organization of nucleic acids and lipids.
A novel labeling approach was applied and used for super-resolution imaging of the E. coli nucleoid. It is based on the incorporation of the modified thymidine analogue 5-ethynyl-2’- deoxyuridine (EdU) into the replicating chromosome. Azide-functionalized organic fluorophores can be covalently attached to the ethynyl group of incorporated EdU bases using a copper-catalyzed "click chemistry" reaction. Under the investigated growth condition, E. coli cells exhibited overlapping replication cycles, which is commonly referred to as multi-fork replication and enables cells to divide faster than they can replicate the entire chromosome. dSTORM imaging of such labeled nucleoids revealed chromosome features with diameters of 50 - 200 nm, representing highly condensed DNA filaments. Sorting single E. coli cells by length allowed visualizing structural changes of the nucleoid throughout the cell cycle. Replicating nucleoids segregated and expanded along the bacterial long axis, while constantly covering the entire width of the cell. Measuring cell and nucleoid length revealed a relative nucleoid expansion rate of 78 ± 6 %. At the same time, nucleoids populated 63 ± 8 % of the cell length, almost exclusively being localized to the cylindrical part of the cell. This value was hence normalized to the cylindrical fraction of the cell, yielding a value of 79 ± 10 % (nucleoid-populated fraction of the cell cylinder), which is in good agreement with the observed relative nucleoid expansion rate. These results therefore support a growth-mediated segregation model, in which the chromosome is anchored to the inner membrane and passively segregated into the prospective daughter cells upon cell growth. 3-dimensional dSTORM imaging of labeled nucleoids confirmed that compacted nucleoids helically wrap along the inner membrane. Similar results were obtained by imaging orthogonally aligned E. coli cells using a holographic optical tweezer approach.
In order to visualize particular proteins together with the nucleoid, several correlative imaging workflows were established, facilitating multi-color SMLM imaging in single E. coli cells. These workflows bypass prior limitations of SMLM, including destruction of FPs by reactive oxygen species in copper-catalyzed click reactions or incompatibility of PALM imaging with dSTORM imaging buffers. A sequential SMLM imaging routine was developed which is based on postlabeling and retrieval of previously imaged cells. Optimal imaging conditions can be maintained for each fluorophore, enabling to extract quantitative information from PALM measurements while correlating the protein distribution to the nucleoid ultrastructure within the highly resolved cell envelope. Applying this workflow to an E. coli strain carrying a chromosomal rpoC - photoactivatable mCherry (PAmCh) fusion, transcribing RNA polymerase (RNAP) was found to be localized on the surface of nucleoids, where active genes are exposed towards the cytosol. During growth in nutrient-rich medium, the majority of RNAP molecules was bound to the chromosome, thus ensuring that the RNAP pool is equally distributed to the daughter cells upon cell division. This work represented the first triple-color SMLM study performed in E. coli cells. ...
Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
(2021)
Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation.
The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation.
In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities.
The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD.
The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A).
The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted.
The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend.
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Die Biosynthese der Fettsäuren (FS) ist in Eukaryoten und Bakterien ein hochkonserviert zentraler Stoffwechselweg, der in zwei strukturell verschiedenen Systemen ausgeführt wird. Die meisten Bakterien, Parasiten, Pflanzen und Mitochondrien nutzen ein Fettsäuresesynthase Typ-II (FAS-II) System. Bei FAS II Systemen sind alle katalytischen Domänen separate lösliche Proteine. In Eukaryoten wie auch den Bakterien Corynebakteria, Mycobakteria, Nocardia (Klasse der CMN Bakterien) liegen die katalytischen Domänen fusioniert auf einer Polypeptidkette vor, die zu einem Multienzymkomplex der Fettsäuresynthase Typ I (FAS-I) assemblieren. Die Architektur der FAS-I zeigt große Unterschiede; die X förmige Säuger-FAS-I (Maier et al., 2006), sowie die fassartigen Enzyme der Pilz FAS-I (Jenni et al., 2007; Leibundgut et al., 2007; Lomakin et al., 2007; Johansson et al., 2008) und der bakteriellen FAS-I (Boehringer et al., 2013; Ciccarelli et al., 2013). Zwischen Pilz- und bakterieller FAS-I gibt es trotz des ähnlichen Aufbaus bedeutende Unterschiede. Mycobakterium tuberculosis, der Auslöser von Tuberkulose (TB), an der jährlich über eine Million Menschen weltweit sterben (WHO, 2014), synthetisiert durch eine Symbiose von FAS-I, FAS-II und der Polyketidsynthase-13 Mykolsäuren. Durch die Mykolsäuren ist M. tuberculosis resistent gegen äußere Einflüsse. FAS-I ist in die Synthese der Vorstufen der Mykolsäuren involviert. Sie stellt im Kampf gegen TB ein potentielles Inhibierungstarget dar.
Strukturell war die bakterielle FAS-I beim Beginn der vorliegenden Arbeit, nur durch negative-stain-Elektronenmikroskopie (EM) Aufnahmen aus dem Jahr 1982 charakterisiert (Morishima et al., 1982). In dieser Arbeit konnte die bakteriellen FAS I aus M. tuberculosis (MtFAS), sowie Corynebacterium ammoniagenes (CaFAS) und Corynebacterium efficiens (CeFAS) strukturell untersucht werden. Dies geschah mit den Methoden negative-stain-EM, Einzelmolekül-Cryo-EM (Cryo-EM), Cryo EM Tomographie (CET) und Röntgenkristallographie.
Anhand von CeFAS-Kristallen konnte erstmals durch Röntgenkristallographie die Struktur einer bakteriellen FAS-I bestimmt werden. Zudem wurde die hohe konformationelle Flexibilität der bakteriellen FAS-I mit mehreren Methoden gezeigt. Für die CaFAS konnte mit Cryo-EM initiale Prozesse der Proteinkristallbildung abgebildet werden.
Xenorhabdus and Photorhabdus bacteria are gaining more and more attention as a subject of research because of their unique yet similar life cycle with nematodes and insects. This work focused on the secondary metabolites that are produced by Xenorhabdus and Photorhabdus. With the help of modern HPLC-MS methodologies and increasingly available bacterial genome sequences, the structures of unknown secondary metabolites could be elucidated and thus their biosynthesis pathways could be proposed, too.
The first paper reported 17 depsipeptides termed xentrivalpeptides produced by the bacterium Xenorhabdus sp. 85816. Xentrivalpeptide A could be isolated from the bacterial culture as the main component. The structure of xentrivalpeptide A was elucidated by NMR and the Marfey´s method. The remaining xentrivalpeptides were exclusively identified by feeding experiments and MS fragmentation patterns.
The second paper described the discovery and isolation of xenoamicin A from Xenorhabdus mauleonii DSM17908. Additionally, other xenoamicin derivatives from Xenorhabdus doucetiae DSM17909 were analyzed by means of feeding experiments and MS fragmentation patterns. The xenoamicin biosynthesis gene cluster was identified in Xenorhabdus doucetiae DSM17909.
The manuscript for publication focused on the biosynthesis of anthraquinones in Photorhabdus luminescens. The Type II polyketide synthase for the biosynthesis of anthraquinone derivatives was discovered in P. luminescens in a previous publication by the Bode group,1 in which a partial reaction mechanism for the biosynthesis has been proposed. The manuscript reported in this thesis however elucidated the biosynthetic mechanisms in a greater detail as compared to the previous publication. Particularly, the biosynthetic mechanism was deciphered through heterologous expression of anthraquinone biosynthesis (ant) genes in E. coli. Additionally, deactivation of the genes antG encoding a putative CoA ligase and antI encoding a putative hydrolase, was performed in P. luminescens. Selected ant genes were over-expressed in E. coli as well as the corresponding proteins purified for in vitro assays. Model compounds were chemically synthesized as possible substrates of AntI and were used for in vitro assays. Here, it was revealed that the CoA ligase AntG played an essential role in the activation of the ACP AntF. Furthermore, a chain shortening mechanism by the hydrolase AntI was identified and was further confirmed by in vitro assays using model compounds. Additionally, this chain shortening mechanism was supported by homology based structural modeling of AntI.
Die hier vorliegende Dissertation befasst sich mit der Synthese von Naturstoffen aus Xenorhabdus und Photorhabdus spp. Da 6,0 - 7,5% ihres Genoms Sekundärmetabolit Clustern zuzuordnen sind, gelten diese entomopathogenen Bakterien als vielversprechende Naturstoffproduzenten. Die Palette der von ihnen produzierten Naturstoffe reicht von Antibiotika über Insektizide bis hin zu potentiellen Zytostatika. Die im Rahmen dieser Arbeit synthetisierten und charakterisierten Substanzen lassen sich in vier Kategorien einteilen: kleine Sekundärmetabolite (Phurealipide), zyklische Makrolaktame (Xenotetrapeptide, GameXPeptide und Ambactin), zyklische Makrolaktone (Szentiamide, Xentrivalpeptide und Xenephematide) und methylierte lineare Peptide (Rhabdopeptide und Rhabdopeptid-ähnliche Moleküle).
Development and implementation of novel optogenetic tools in the nematode Caenorhabditis elegans
(2016)
Optogenetics, though still only a decade old field, has revolutionized research in neurobiology. It comprises of methods that allow control of neural activity by light in a minimally-invasive, spatio-temporally precise and genetically targeted manner. The optogenetic actuators or the genetically encoded light sensitive elements mediate light driven manipulation of membrane potential, intracellular signalling, neuronal network activity and behaviour (Fenno et al. 2011; Dugué et al. 2012). These techniques have been particularly useful for dissecting neural circuits and behaviour in the transparent and genetically amenable nematode model system Caenorhabditis elegans (Husson et al. 2013; Fang-yen et al. 2015).
In fact, C. elegans was the first living organism in which microbial rhodopsin based optogenetic tools (Channelrhodopsin-2 or ChR2, and Halorhodopsin or NpHR) were successfully implemented and bimodal 'remote' control of behaviour was achieved (Nagel et al. 2005; Zhang et al. 2007). Since then it has been a prominent model for the development and application of novel optogenetic tools and techniques, especially in the nervous system which comprises of 302 neurons and is organised in a hierarchical organization. The environmental stimuli are sensed by the sensory neurons, leading to the processing of information by the downstream interneurons, that relay to motor neurons which in-turn synapse onto muscles that drive the movement-based responses.
The microbial rhodopsins like ChR2 and NpHR mediate light driven depolarization and hyperpolarization, respectively and thereby activate or inhibit neural activity. However, they do not allow local control of membrane potential as they are expressed all over the plasma membrane of the cell rather than being restricted to specific domains, for example synaptic sites. Moreover, they completely over-ride the intrinsic activity of the cell, completely bypassing the signal transduction processes inside the cell. Thus, in order to study intracellular signalling and to answer questions pertaining to the endogenous role of receptors and channels in an in-vivo context, the optogenetic tool-kit needs to be expanded.
This thesis aimed at developing and implementing novel optogenetic tools in C. elegans that allow for sub-cellular signalling control as well as endogenous receptor control. These are: two light activated guanylyl cyclases (bPGC and BeCyclOp) to modify cyclic guanosine monophosphate (cGMP) mediated signalling in the sensory neurons, as well as attempts towards rendering endogenous C. elegans receptors - glutamate receptor (GLR-3/-6), acetylcholine receptor (ACR-16), glutamate gated chloride channel (GLC-1) light switchable and to understand their biological function in-vivo.
Organisms respond to sensory cues by activation of a primary receptor followed by relay of information downstream to effector targets by secondary signalling molecules. cGMP is a widely used 2nd messenger in cellular signaling, acting via protein kinase G or cyclic nucleotide gated (CNG) channels. In sensory neurons, cGMP allows for signal modulation and amplification, before depolarization. Chemo-, thermo-, and oxygen-sensation in C. elegans involve sensory neurons that use cGMP as the main 2nd messenger. For example, ASJ is the pheromone sensing neuron regulating larval development, AWC is the chemosensory neuron responding to volatile odours and BAG senses oxygen and carbon dioxide in the environment. In these neurons, cGMP acts downstream of the GPCRs and functions by activating cationic TAX-2/-4 CNG channels, thereby depolarising the sensory neuron. Manipulating cGMP levels is required to access signalling between sensation and sensory neuron depolarization, thereby provide insights into signal encoding. We achieve this by implementing two photo-activatable guanylyl cyclases - 1) a mutated version of Beggiatoa sp. bacterial light-activated adenylyl cyclase, with specificity for GTP (Ryu et al. 2010), termed BlgC or bPGC (Beggiatoa photoactivated guanylyl cyclase) and 2) guanylyl cyclase rhodopsin (Avelar et al. 2014) from Blastocladiella emersonii (BeCyclOp).
bPGC is a BLUF (blue light sensing using flavin) domain containing cyclase which uses FAD as the co-factor and catalyses the synthesis of cGMP from GTP upon activation by blue light. Prior to implementation in sensory neurons, a simpler heterologous system with co-expression of the TAX-2/-4 CNG channel in C. elegans body wall muscle (BWM) was used. The cGMP generated by the light activated cyclases activates the CNG channel leading to the muscle depolarization, thereby causing changes in body length which can be easily scored.
The baker’s yeast Saccharomyces cerevisiae is a valuable and increasingly important microorganism for industrial applications (Hong and Nielsen, 2012). Its robustness concerning process conditions like low pH, osmotic and mechanical stress as well as toxic compounds is an advantage. Moreover, S. cerevisiae is ‘generally regarded as safe’ (GRAS). The model organism has been studied intensively. The collected data, including genomic, proteomic and metabolic information, can be used to genetically modify and improve its metabolism. Fatty acids and fatty acid derivatives have wide applications as biofuels, biomaterials, and other biochemicals. Several studies have been dealing with the overproduction of fatty acids and derivatives thereof in S. cerevisiae. The fatty acid biosynthesis starting with acetyl-CoA requires two enzymes, the acetyl-CoA carboxylase (Acc1p) and the fatty acid synthase complex (FAS), to produce acyl-CoA esters with predominantly 16 to 18 carbon atoms chain length (Lynen et al., 1980). For the synthesis of monounsaturated fatty acids in S. cerevisiae the ER bound acyl-CoA desaturase, Ole1p is essential (Tamura et al., 1976; Certik and Shimizu, 1999).
Using S. cerevisiae, the first section of this work dealt with the heterologous characterization of potential ω1-desaturases. Due to the fact that unsaturated fatty compounds can be modified further by hydrosilylations, hydrovinylations, oxidations to epoxides, acids, aldehydes, ketones or metathesis reactions, the interest in ω1-fatty acids is tremendous (Behr and Gomes, 2010). With the intention to find enzymes in fungi, that have a terminal desaturase activity a search in different genome databases was performed. The sequences of Pex-Desat3 and Obr-TerDes were used as reference sequences. The analysed proteins from Schizophyllum commune (EFI94599.1), Schizosaccharomyces octosporus (EPX72095.1), Wallemia mellicola (EIM20316.1), Wallemia ichthyophaga (EOR00207.1) and Agaricus bisporus var. bisporus (EKV44635.1), however, finally turned out to be Δ9 desaturases. A fungal desaturase with ω1-activity could not be found. The Δ9 desaturase SCD1 from Mus musculus was crystallized by Bai et al. (2015) and the information for specific amino acids responsible for the substrate specificity or enzyme activity were allocated. In combination with sequence and enzyme activity data form ChDes1 from Calanus hyperboreus, Desat2 from Drosophila melanogaster, Pex-Desat3 from Planotortrix excessana and Obr-TerDes from Operophtera brumata single amino acid exchanges were performed in the Δ9 desaturase Ole1p from S. cerevisiae. For all mutants, only fatty acids (C16 - C18) with a double bond between carbon C9 and C10 could be found. This indicates, that all inserted amino acid exchanges do not affect the substrate specificity or the position of the introduced double bond.
In the second section the focus was in the development of a production system for fatty acids in S. cerevisiae with regard to the previously established procedures by metabolic engineering. The combination of cytosolic malate dehydrogenase (MDH3), cytosolic malate enzyme (MAE1) and a citrate- α-ketoglutarate- carrier (YHM2) should improve the availability of acetyl-CoA in the cytosol, which is an important precursor for the fatty acid biosynthesis. If the major pathway (acetyl-CoA carboxylase and fatty acid synthase) was already optimized by high expression levels than no positive effect on increased fatty acid synthesis was detectable. Only non-optimized strains, with the additional overexpression of ATP-citrate lyase and cytosolic malate dehydrogenase, lead to a 41 % (20 mg/g dcw) improvement of fatty acid synthesis. In order to increase the fatty acid content further, the additional overexpression of DGA1 and TGL3 was performed. Hence, the highest amount of fatty acids could be observed with the strain S. cerevisiae WRY1ΔFAA1ΔFAA4 (2.5 g/L ± 0.8 g/L). The additional elimination of acyl-CoA synthetase Fat1p did not improve the yield.
It was recently reported, that chain length control of the fatty acid synthesis of bacterial FAS can be changed by rational engineering (Gajewski et al., 2017a). The knowledge about bacterial FAS was transferred in this work to S. cerevisiae FAS. Mutating up to five amino acids in the FAS complex enabled S. cerevisiae to produce medium chain fatty acids (C6 - C12). Further improvement was done by metabolic pathway engineering (promoter of alcohol dehydrogenase II from S. cerevisiae (pADH2), deletion of acyl-CoA synthetase FAA2) and optimization of fermentation conditions (YEPD-bacto medium buffered with potassium phosphate). The production of medium chain fatty acids resulted in the highest yield of 464 mg/L (C6 to C12 fatty acids). Furthermore, strains were created specifically overproducing hexanoic acid (158 mg/L) and octanoic acid (301 mg/L). The characterization of transferases, which could be responsible for the de-esterification of CoA-bound fatty acids, was analysed in an additional approach. It could be shown, that the genes EHT1, EEB1 and MGL2 have an influence on the MCFA yield in the supernatant. Generally speaking, the data from the single and double deletion strains suggest that Eeb1p has a selective hydrolytic activity for hexanoic acid-CoA ester, while Eht1p shows selective hydrolytic activity for octanoic acid-CoA ester, which is in line with Saerens et al. (2006).
Um molekulare Mechanismen in biologischen Prozessen zu verstehen, ist es unerlässlich biologisch aktive Verbindungen zu kontrollieren. Dabei spielt besonders die Aktivierung bzw. Desaktivierung von Genabschnitten eine zentrale Rolle in der gegenwärtigen chemischen, biologischen und medizinischen Forschung. Nukleinsäuren sind dabei offenkundige Zielmoleküle, da sie die Genexpression auf unterster Ebene regulieren und auf vielfältige Art und Weise an biologischen Prozessen beteiligt sind. Um solch eine genaue Steuerung zu erreichen, werden Nukleinsäuren häufig photolabil modifiziert und unter die Kontrolle von Licht gebracht. Da hochentwickelte Technologien es erlauben Photonen bestimmter Energie unter präziser räumlicher und zeitlicher Auflösung zu dosieren, ist Licht als nicht invasives Triggersignal ein besonders geeignetes Werkzeug um molekulare Prozesse zu kontrollieren.
Die Verwendung photolabiler Schutzgruppen („cage“) ermöglicht es, diese lichtaktivierbaren Nukleinsäuren („caged compound“) herzustellen. Üblicherweise werden Oligonukleotide damit an funktionsbestimmenden Stellen versehen, woraufhin die Funktion der Oligonukleotide unterdrückt wird. Die biologische Aktivität kann durch Bestrahlung mit Licht wieder hergestellt werden, da die photolabile Schutzgruppe durch den Lichtimpuls abgespalten wird. Neben der zeitweiligen Maskierung der Nukleinsäureaktivität existiert auch eine Methode, die als „photoaktivierbarer Strangbruch“ (‘‘caged strand break‘‘) bezeichnet wird. Dabei werden mit Hilfe von photolabilen Linkern (‘‘Verknüpfer‘‘) lichtinduzierte Strangbrüche in Oligonukleotiden ausgelöst, um so beispielsweise die Struktur eines Nukleinsäurestrangs zu zerstören. Die Idee der photoaktivierbaren Strangbrüche ist nicht neu, dennoch werden photolabile Schutzgruppen überwiegend nach der erstgenannten Strategie verwendet. Im Rahmen dieses Promotionsvorhabens wurden neue photosensitive Linkerbausteine für Oligonukleotide entwickelt und hergestellt, welche sich vor allem im Hinblick auf die Anwendbarkeit in lebenden biologischen Systemen von den bisherigen photolabilen Linkern unterscheiden.
Im ersten Projekt wurde ein nicht-nukleosidischer, photolabiler Linker, basierend auf dem Cumaringrundgerüst, entwickelt. Das Ziel war hier, vor allem, einen zweiphotonenaktiven Linker für biologische Anwendungen und Zweiphotonen-Fragestellungen nutzbar zu machen. Bisherige Zweiphotonen-Linker konnten hauptsächlich nur für Proteinverknüpfungen bzw. Neurotransmitter verwendet werden oder mussten chemisch umständlich (z.B. Click-Chemie) und postsynthetisch in Oligonukleotide eingeführt werden. Der neu entwickelte Zweiphotonen-Linker wurde als Phosphoramiditbaustein für die Oligonukelotid-Festphasensynthese synthetisiert, was einen problemlosen und automatisierten Einbau garantiert. Mit einem modifizierten Oligonukleotid konnten die photochemischen Eigenschaften des Linkers bestimmt und mit Hilfe eines fluoreszenzbasierten Verdrängungsassays und Lasertechniken der Zweiphotonen-Effekt visualisiert werden. Dazu wurde ein Hairpin-DNA-Strang hergestellt, welcher eine Linkermodifikation im Bereich der Loopregion enthält. Durch eine Thiolmodifikation am 5‘-Ende des Oligonukleotidstranges war es möglich, diesen in einem Maleimid-funktionalisierten Hydrogel zu fixieren. Ein DNA-Duplex mit einem Fluorophor/Quencherpaar und einer korrespondierenden Sequenz zum modifizierten Hairpin-Strang wurde ebenfalls dem System zugegeben, allerdings wurde dieser nicht fixiert, um Diffusion zu ermöglichen. Durch die räumliche Nähe des Fluorophors zum Quencher konnte im unbelichteten Zustand zunächst keine Fluoreszenz gemessen werden. Mit einem (Femtosekunden-)gepulsten Laser und dem damit verbundenen Bindungsbruch im Hairpin-Strang durch Zweiphotonen-Effekte wurde es dem fluoreszierenden Strang des DNA-Duplex ermöglicht, sich vom Quencher-Strang zu lösen und an den fixierten Strang zu hybridisieren. Das Photolyse-Ereignis konnte so in ein lokales Fluoreszenzsignal übersetzt und detektiert werden.
Der eindeutige Beweis, dass es sich tatsächlich um ein Zweiphotonen-induziertes Ereignis handelt, konnte durch die dreidimensional aufgelöste Photolyse und über die quadratische Anhängigkeit des Fluoreszenzsignals von der eingestrahlten Laserleistung erbracht werden.
Die generelle Kompatibilität des Cumarin-Linkers mit biologischen Systemen konnte in Zellkulturexperimenten gezeigt werden. Dazu wurde eine Transkriptionsfaktor-DNA Decoy-Strategie entwickelt, in der Linker-modifizierte DNA Decoys an regulatorische Transkriptionsfaktoren binden und diese aber auch photochemisch wieder freisetzen können („catch and release-Strategie“). Zellkulturexperimente, um mit dieser Methode das Transkriptionsfaktor-gesteuerte und endogene Gen für Cyclooxygenase-2 (COX2) zu regulieren, lieferten keine aussagekräftigen Ergebnisse. Daher wurden die verwendeten Zellen dahingehend manipuliert, sodass sie das Protein GFP (grün fluoreszierendes Protein) in Abhängigkeit von der Anwesenheit eines Transkriptionsfaktors exprimieren. Das so durch die Zellen verursachte Fluoreszenzsignal steht in direkter Abhängigkeit zur Decoy-Aktivität. Mit Hilfe modifizierter GFP-Decoys konnte hierbei eine Regulation auf Transkriptionsebene in biologischen Organismen erreicht werden. Mit dem Electrophoretic Mobility Shift Assay (EMSA), einer molekularbiologischen in vitro-Analysetechnik, wurden die Interaktionen zwischen modifizierten Decoys und dem Transkriptionsfaktor untersucht.
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The composition of cellular membranes is extremely complex and the mechanisms underlying their homeostasis are poorly understood. Organelles within a eukaryotic cell require a non-random distribution of membrane lipids and a tight regulation of the membrane lipid composition is a prerequisite for the maintenance of specific organellar functions. Physical membrane properties such as bilayer thickness, lipid packing density and surface charge are governed by the lipid composition and change gradually from the early to the late secretory pathway. As the endoplasmic reticulum (ER) is situated at the beginning of the cells secretory pathway, it has to accept and accommodate a great variety and quantity of secretory and transmembrane proteins, which enter the ER on their way to their final cellular destination. Secretory proteins can be translocated into the lumen of the ER co- or posttanslationally and membrane proteins are being inserted and released into the ER membrane. In the oxidative milieu of the ER-lumen, supported by a variety of chaperones, proteins can fold into their native form.
If the folding capacity of the ER-lumen is exceeded, an accumulation of mis- or unfolded proteins in the lumen of the ER occurs, consequently triggering the unfolded protein response (UPR). This highly conserved program activates a wide-spread transcriptional response to restore protein folding homeostasis. In fact, 7 – 8% of all genes in the yeast Saccharomyces cerevisiae (S. cerevisiae) are regulated by the UPR. The mechanism underlying the activation of the UPR by protein folding stress has been investigated thoroughly in the last decades and many of its mechanistic details have been elucidated. Recently, it became evident that aberrant lipid compositions of the ER membrane, collectively referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism of this membrane-activated UPR, however, remained unclear.
This study focuses on the UPR in S. cerevisiae and characterizes the inositol requiring enzyme 1 (Ire1) as the sole UPR sensor in S. cerevisiae. Active Ire1 forms oligomers and, collaboratively with the tRNA ligase Rlg1, splices immature mRNA of the transcription factor HAC1, which results in the synthesis of mature HAC1 mRNA and the production of the active Hac1 protein, which binds to UPR-elements in the nucleus and activates the expression of UPR target genes. Here, the combination of in vivo and in vitro experiments is being used, which is supplemented by molecular dynamics (MD) simulations performed by Roberto Covino and Gerhard Hummer (MPI for Biophysics, Frankfurt), aiming to identify the molecular mechanism of Ire1 activation by lipid bilayer stress. This study focuses on the analysis of the juxta- and transmembrane region of Ire1. Bioinformatic analyses revealed a putative ER-lumenal amphipathic helix (AH) N-terminally of and partially overlapping with the transmembrane helix (TMH). This predicted AH contains a large hydrophobic face, which inserts into the ER membrane, forcing the TMH into a tilted orientation within the membrane. The resulting unusual architecture of Ire1’s AH and TMH constitutes a unique structural element required for the activation of Ire1 by lipid bilayer stress.
To investigate the function of the AH in the physiological context, different variants of Ire1 were produced under the control of their endogenous promoter and from their endogenous locus. The functional role of the AH was tested, by disrupting its amphipathic character by the introduction of charged residues into the hydrophobic face of the AH. The role of a conserved negative residue between the TMH and the AH (E540 in S. cerevisiae) was tested by substituting it by a unipolar, polar, or positively charged residue. These variants were intensively characterized using a series of assays:
This thesis provides evidence that the AH is crucial for the function of Ire1: Mutant variants with a disrupted (F531R, V535R) or otherwise modified AH (E540A) exhibited a lower degree of oligomerization and failed to catalyze the splicing of the HAC1 mRNA as the Wildtype control. Likewise, the induction of PDI1, a target gene of the UPR, was greatly reduced in mutants with a disrupted or defective AH. These data revealed an important functional role of the AH for normal Ire1 function.
An in vitro system was established to analyze the membrane-mediated oligomerization of Ire1. This system enabled the isolated functional analysis of the AH and TMH during Ire1 activation by lipid bilayer stress. A fusion construct, coding for the maltose binding protein (MBP) from Escherichia coli (E. coli), N-terminally to the AH and TMH of Ire1 was produced. The heterologous production in E. coli, the purification and reconstitution of this minimal sensor of Ire1 in liposomes was established as part of this study. To analyze the oligomeric status of the minimal sensor in different lipid environments, continuous wave electron paramagnetic resonance (cwEPR) spectroscopic experiments were performed. These experiments revealed that the molecular packing density of the lipids had a significant influence of the oligomerization of the spin-labeled membrane sensor: increasing packing densities resulted in sensor oligomerization. The AH-disruptive F531R mutant, in which the amphipathic character of the AH was destroyed, showed no membrane-sensitive changes in its oligomerization status.
Thus, the activation of Ire1 by lipid bilayer stress is achieved by a membrane-based mechanism. According to the current model, the AH induces a local membrane compression by inserting its large hydrophobic face into the membrane. As membrane thickness and acyl chain order are interconnected, this compression simultaneously results in an increased local disordering of lipid acyl chains. Supporting MD simulations performed by Roberto Covino and Gerhard Hummer revealed that the bilayer compression is significantly more pronounced in a densely packed lipid environment, than in a lipid environment of lower lipid packing density. Hence, the energetic cost of the local compression increases with the packing density of the membrane, but is compensated for by the oligomerization of Ire1. This minimization of energetic cost induced by the membrane deformation of Ire1 forms the basis for the activation of Ire1 by lipid bilayer stress.
Fettsäuresynthasen vom Typ I (FAS I), hier bezeichnet als Fettsäuremegasynthasen,sind Multienzymkomplexe, in denen sämtliche funktionellen Domänen für die de-novo-Synthese von Fettsäuren einen strukturellen Verbund eingehen. Auch das für den Transport von Edukten und Intermediaten nötige Acyl Carrier Protein (ACP) ist kovalent gebundener Teil dieses Komplexes, der so zu einer hocheffizienten molekularen Maschine zur Massenproduktion dieser grundlegend essentiellen Zellbausteine wird. Die FAS I aus Pilzen (fFAS), als Gegenstand dieser Arbeit, mit einer Masse von bis zu 2,7 MDa ist heute in ihrer Struktur durch Röntgenkristallographische sowie elektronenmikroskopische Methoden gut charakterisiert. 48 funktionelle Domänen sind zu einem geschlossenen Reaktionskörper angeordnet, indem sie in einer strukturgebenden Matrix aus Expansionen und Insertionen bzgl. der enzymatischen Kerndomänen eingebettet sind, die 50% des gesamten Proteins ausmacht. Neben den zahlreichen strukturellen Informationen über fFAS ist jedoch noch wenig über ihre Assemblierung verstanden. Dabei ist sie nicht nur als ein Beispiel für das generelle Verständnis von Assemblierungsmechanismen von Multienzymkomplexen interessant, sondern wird hier auch als Ziel eines inhibitorischen Eingriffs betrachtet, um eine neue antimykotische Wirkstrategie abseits des Ausschaltens aktiver Zentren zu evaluieren. Nur wenn die Mechanismen und Wechselwirkungen im Assemblierungsprozess offen gelegt sind, lassen sie sich später gezielt attackieren. Essentielle Sekundärstrukturmotive müssen identifiziert und bewertet werden, um sie einer weiteren Evaluation als Drug-Target-Kandidaten zugänglich zu machen. In dieser Arbeit werden Resultate aus in-vivo-Experimenten an rational mutierten fFAS-Konstrukten unter Zuhilfenahme einer evolutionären Betrachtung der fFAS gemeinsam mit Erkenntnissen aus andernorts geleisteten in-vitro-Experimenten an fFAS-Fragmenten zu einem geordneten Assemblierungsweg der fFAS zusammengeführt. Dabei werden Evidenzen aus den Kausaltäten zentraler Anforderungen an einen Assemblierungsmechanismus der fFAS zu drei konsequenten Schlüsselschritten verdichtet, die (i) eine frühe Interaktion zweier komplementärer Polypeptidketten zu einer Pseudo-Einzelkette, (ii) eine posttranslationale Modifikation von ACP und (iii) die geordnete Reifung zum fertigen Komplex durch Selbstassemblierung der beteiligten Domänen umfassen. Durch rationale Mutationen an den Schnittstellenmotiven für die Pseudo-Einzelkettenbildung, werden diese als Schwachstelle der Assemblierung unterschiedlicher fFAS-Typen charakterisiert, wobei für S. cerevisiae nicht weniger als zwei gezielte Punktmutationen ausreichen, um die Assemblierung des gesamten Komplexes zu verhindern. Darüber hinaus zeigen Experimente mit fFAS-Konstrukten, deren Schnittstellenmotive einer intramolekular kompetitiven Wechselwirkung ausgesetzt sind, prinzipiell die Möglichkeit zur Inhibierung der fFAS-Assemblierung durch Störung der Pseudo-Einzelkettenbildung.
If the biotechnological production of chemicals can further replace or support regular synthetic chemistry, industry will be able to move away from fossil oils towards renewable sources. However, in many cases the much needed adaptation of biotechnological production systems is not yet developed to the necessary level.
For processes where short fatty acids (FA) are needed, as for example in the microbial production of biofuels in the gasoline range, protein engineering had not yet delivered feasible solutions. In this thesis, several approaches to introduce chain length control on type I fatty acid synthases (FAS) were established and made available in a publication and two patents. Therein, engineering was focused on rational design based on available structural information.
First, the type I FAS from C. ammoniagenes was used as a model enzyme to probe modifications on FAS in a low complex in vitro environment in order to gain information about structure-function relationships. At this stage, engineering was conducted in several rounds, first addressing possible ways to alter product distributions by changing substrate affinities through concise mutations in binding channels. Several FAS constructs were generated ranging from first successes, where short FA were produced as side products, to FAS where native chain length programming was overwritten and only short FA were produced.
Furthermore, another engineering target was addressed with the modification of domain-domain interactions on FAS. For its exploitation to direct synthesis, contact surfaces on catalytic domains were changed to interfere with acyl carrier protein binding. This channeling of the kinetic process on the enzyme led to similar successes and short FA became the primary product.
The two approaches have proven to be potent tools to introduce systems of chain length control in FAS. This rational engineering has the big advantage that it is mostly minimally invasive and due to the high conservation of de novo FA synthesis, individual mutations could easily be used in other FAS (and their organisms) as well. Even heterologous expression of modified FAS genes is feasible.
Engineering was not only tested in a defined in vitro environment and but also in S. cerevisiae as an exemplary in vivo system. The results eventually confirmed the in vitro findings and proved that the chosen engineering could be transferred to more complex systems. Even before any optimization for highest output, the titers of short FA from S. cerevisiae fermentation matched previous reports with 118 mg/L.
In sum, this work covers several layers from basic research to preliminary applications. The presented modifications to create short FA producing FAS can be a key step in synthesis pathways and will likely enable a whole range of new succeeding research. It can be seen as a valuable contribution towards establishing novel ways for the production of chemicals from renewable sources.
Natural products are valuable sources for biologically active compounds, which can be utilized as pharmaceuticals. Thereby, the synthesis is based purely on biosynthetic grounds often conducted by so-called megaenzymes. One major biosynthetic pathway is the acetate pathway including polyketide and fatty acid synthesis, which encompass one of the largest classes of chemically diverse natural products. These have medicinal relevance due to their antibacterial, antifungal, anthelmintic, immunosuppressive and antitumor properties.
Due to the high structural and functional similarity between polyketide synthases and type I animal fatty acid synthases (FASs), FAS can serve as a paradigm for the whole class of multifunctional enzymes. To fully exploit the biosynthetic potential of FASs, a good access to the enzyme is of essential importance. In this regard, Escherichia coli remains an unchallenged heterologous host due to low culturing costs, particularly fast mutagenesis cycles and relatively easy handling. Surprisingly, no sufficient expression strategy for an animal FAS in E. coli has yet been reported, as it turned out that the only approach was not reproducible.
We commenced our analysis with searching for an appropriate FAS homolog that fulfills our requirements of high protein quality, sufficient yield and ensured functionality. After extensive screening of different variants, culturing conditions and co-expression strategies, we identified the murine FAS (mFAS) as our protein of choice. The established purification strategy using tags at both termini led to a reproducible and sufficient access to the protein in excellent quality. The enzyme was further biochemically characterized including an enzyme kinetic investigation of fatty acid synthesis and an examination whether different acyl-CoA substrates can serve as priming units. This adds mFAS to our repertoire of manageable megaenzymes paving the way to exploit the catalytic efficiency in regards of microbial custom-compound synthesis.
With a strong focus on deepening our understanding of the working mode of such megaenzymes, rather than analyzing respective biosynthetic products, we have addressed the question whether mFAS itself can be engineered towards PKSs or whether properties of mFAS can be exploited to engineer PKSs. This approach was conducted on three levels of complexity from function of individual domains via organization of domains to form modules to the interplay of two modules in bimodular constructs.
Fatty acid synthesis begins with the loading of acyl moieties onto the FAS, which is conducted by a domain called malonyl-/acetyltransferase (MAT). This domain was in-depth characterized due to its important role of choosing the substrates that are built in the final compound. Our analysis comprised structural and functional aspects providing crystal structures of two different acyl-bound states and kinetic parameters for the hydrolysis and transacylation reaction using twelve exemplary CoA-esters. For this purpose, we have successfully established a continuous fluorometric assay using the α-ketoglutarate dehydrogenase as a coupled enzyme, which converts the liberated coenzyme A into Nicotinamide adenine dinucleotide. These data revealed an extensive substrate ambiguity of the MAT domain, which had not been reported to that extent before. Further, we could demonstrate that the fold fulfills both criteria for the evolvability of an enzyme by expressing MAT in different structural arrangements (robustness) and by altering the substrate ambiguity within a mutagenesis study (plasticity). Taken these aspects together, we are persuaded that the MAT domain can serve as a versatile tool for PKSs engineering in potential FAS/PKS hybrid systems.
On the higher level of complexity, we investigated the architectural variability of the mFAS fold, which constitutes a fundamental basis for a broader biosynthetic application. We could rebuild all four module types occurring in typical modular PKSs confirming a high degree of modularity within the fold. Not only structural, but also functional integrity of these modules was validated by using triacetic acid lactone formation and ketoreductase activity. Especially the latter analysis, made it possible to quantify effects of the engineering within the processing part by respective enzyme kinetic parameters. Expanding our focus beyond a singular module, we have utilized the mFAS fold for designing up to 380 kDa large bimodular constructs. In this approach, a loading didomain was attached N-terminally containing an additional MAT and acyl carrier protein (ACP) domain. Two constructs could be expressed and purified in excellent quality to investigate the influence of an altered overall architecture on fatty acid synthesis. By comparison with appropriate controls, a functional effect of the additional loading module could indeed be proven in the bimodular systems. Those constructs allow a comprehensive analysis of the underlying molecular mechanism in the future and serve as a potential model system to study the transition from iterative to vectorial polyketide synthesis in vitro.
The deubiquitinase USP32 regulates non-proteolytic ubiquitination in the endosomal-lysosomal system
(2021)
The regulation of essential cellular processes requires tightly controlled and directed transport of proteins and membranes. The highly dynamic endosomal and lysosomal system forms the key network for exchange and trafficking of molecules with its early endosomes, recycling endosomes, late endosomes, lysosomes, and additionally autophagosomes.
In this system, the small GTPase Rab7 has an essential role at the late endosomal stage regulating vesicle transport, tethering, and fusion, and retromer mediated receptor recycling back to the trans-Golgi network (TGN). Thus, Rab7 is also important for autophagosomes and lysosomes.
Lysosomes do not only represent the end point of the degradation pathway with several feeder pathways. But these organelles are also a dynamic signaling hub for a variety of metabolic processes. The ever-important regulator of cellular biosynthetic pathways mTORC1 dynamically associates with lysosomes where it is activated. mTORC1 activation is a complex multi-step process where a series of signaling events converge in dependence of amino acid levels thereby enabling interactions between the lysosomal v-ATPase, Ragulator complex (consisting of LAMTOR1-5), and Rag GTPases.
Ubiquitin signals are involved in almost all cellular processes. With this, their regulatory mechanism is also described for the endosomal-lysosomal system as well as mTORC1 signaling. Deubiquitinases (DUBs) release conjugated ubiquitin from proteins and thereby maintain the dynamic state of the cellular ubiquitinome.
The ubiquitin-specific protease 32 (USP32) is a poorly characterized DUB with only emerging cellular function. However, its predicted domain structure includes two unique domains within the entire DUB family. It has been linked to the development of breast cancer and small cell lung cancer. Furthermore, overexpressed GFP-USP32 was localized at the TGN, and a global mass spectrometry-based DUB interactome study suggested an interaction with the retromer complex. Based on these data, USP32 was a very interesting candidate to study its cellular function in this PhD project.
To investigate the function without disease background, a polyclonal USP32 knockout (USP32KO) RPE1 cell line was generated using the CRISPR/Cas9 technology. First experiments revealed different protein expression levels in various cell lines, and a subcellular localization of USP32 at membranes of the Golgi and lysosomal compartments. In a subsequent SILAC-based ubiquitinome analysis potential substrates of USP32 were identified. Interestingly, various proteins of the endosomal-lysosomal system were detected with enriched non-proteolytic ubiquitination upon USP32 depletion.
The further characterization of Rab7 as USP32 substrate confirmed the USP32-sensitive ubiquitination of Rab7 at lysine (K) residues 191 and 194. The ubiquitination in USP32KO cells did not change the subcellular localization of Rab7, but enhanced the interaction with the effector protein RILP. This implied that Rab7 was either more active or RILP had higher affinity to ubiquitinated Rab7. The subsequent results verified this theory. The retromer mediated recycling of CI-M6PR back to the TGN was faster or more efficient in USP32-depleted cells.
Accompanying this, levels of hydrolases were enriched in lysosomes isolated from USP32KO cells. Notably, USP32 had no direct effect on expression level or assembly of the retromer complex itself.
The observed lysosomal phenotypes connected another identified substrate to the function of USP32 in the endosomal-lysosomal system: LAMTOR1. LAMTOR1 is a component of the Ragulator complex and thus involved in the activation of mTORC1 at the lysosomal surface. Similar as for Rab7, the first experiments to characterize LAMTOR1 as USP32 substrate confirmed the USP32-sensitive ubiquitination at K20 independent of amino acid availability. However, ubiquitination of LAMTOR1 decreased its lysosomal localization in untreated and amino acid starved USP32KO cells. The following label-free interactome study detected a reduced interaction of LAMTOR1 and subunits of the lysosomal v-ATPase upon loss of USP32. This resulted in a shifted subcellular localization of mTOR (subunit of mTORC1) away from lysosomes. Furthermore, direct substrates of mTORC1 were less or slower re-phosphorylated after long amino acid starvation and re-activation of mTORC1 in USP32KO cells indicating a reduced mTORC1 activity.
Both USP32-dependent regulations of Rab7 and LAMTOR1/Ragulator converged in enhanced autophagic processes analyzed by increased LC3 levels upon amino acid starvation and USP32 depletion.
In summary, the presented thesis described the diverse role of USP32 in the endosomal and lysosomal system, and contributes to the understanding of novel ubiquitin signals in this context.
The application of natural products (NPs) as drugs and lead compounds has greatly improved human health over the past few decades. Despite their success, we still need to find new NPs that can be used as drugs to combat increasing drug resistance via new modes of action and to develop safer treatments with less side effects.
Entomopathogenic bacteria of Xenorhabdus and Photorhabdus that live in mutualistic symbiosis with nematodes are considered as promising producers of NPs, since more than 6.5% of their genomes are assigned to biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites. The investigation on NPs from Xenorhabdus and Photorhabdus can not only provide new compounds for drug discovery but also help to understand the biochemical basis involved in mutualistic and pathogenic symbiosis of bacteria, nematode host and insect prey.
Nonribosomal peptides (NRPs) are a large class of NPs that are mainly found in bacteria and fungi. They are biosynthesized by nonribosomal peptide synthetases (NRPSs) and display diverse functions, representing more than 20 clinically used drugs. Although a large number of NRPs have been identified in Xenorhabdus and Photorhabdus, the advanced genome sequencing and bioinformatic analysis indicate that these bacteria still have many unknown NRPS-encoding gene clusters for NRP production that are worth to explore. Therefore, this thesis focuses on the discovery, biosynthesis, structure identification, and biological functions of new NRPs from Xenorhabdus and Photorhabdus.
The first publication describes the isolation and structure elucidation of seven new rhabdopeptide/xenortide-like peptides (RXPs) from X. innexi, incorporating putrescine or ammonia as the C-terminal amines. Bioactivity testing of these RXPs revealed potent antiprotozoal activity against the causative agents of sleeping sickness (Trypanosoma brucei rhodesiense) and malaria (Plasmodium falciparum), making them the most active RXP derivatives known to date. Biosynthetically, the initial NRPS module InxA might act iteratively with a flexible methyltransferase activity to catalyze the incorporation of the first five or six N-methylvaline/valine to these peptides.
The second publication focuses on the structure elucidation of seven unusual methionine-containing RXPs that were found as minor products in E. coli carrying the BGC kj12ABC from Xenorhabdus KJ12.1. To confirm the proposed structures from detailed HPLC-MS analysis, a solid-phase peptide synthesis (SPPS) method was developed for the synthesis of these partially methylated RXPs. These RXPs also exhibited good effects against T. brucei rhodesiense and P. falciparum, suggesting RXPs might play a role in protecting insect cadaver from soil-living protozoa to support the symbiosis with nematodes.
The third publication presents the identification of a new peptide library, named photohexapeptide library, which occurred after the biosynthetic gene phpS was activated in P. asymbiotica PB68.1 via promoter exchange. The chemical diversity of the photohexapeptides results from unusual promiscuous specificity of five out of six adenylation (A) domains being an excellent example of how to create compound libraries in nature. Furthermore, photohexapeptides enrich the family of the rare linear D-/L-peptide NPs.
The fourth publication concentrates on the structure elucidation of a new cyclohexapeptide, termed photoditritide, which was produced by P. temperata Meg1 after the biosynthetic gene pdtS was activated via promoter exchange. Photoditritide so far is the only example of a peptide from entomopathogenic bacteria that contains the uncommon amino acid homoarginine. The potent antimicrobial activity of photoditritide against Micrococcus luteus implies that photoditritide can protect the insect cadaver from food competitor bacteria in the complex life cycle of nematode and bacteria.
The last publication reports a new family of cyclic lipopeptides (CLPs), named phototemtides, which were obtained after the BGC pttABC from P. temperata Meg1 was heterologously expressed in E. coli. The gene pttA encodes an MbtH protein that was required for the biosynthesis of phototemtides in E. coli. To determine the absolute configurations of the hydroxy fatty acids, a total synthesis of the major compound phototemtide A was performed. Although the antimalarial activity of phototemtide A is only weak, it might be a starting point towards a selective P. falciparum compound, as it shows no activity against any other tested organisms.
Multidomain enzymes, such as fatty acid synthases (FASs) or polyketide synthases (PKSs), play a crucial role in the biosynthesis of important natural products. They have a high significance in the development of new pharmaceuticals and various research approaches focus on the engineering of these proteins. For example, human type I FAS is an interesting therapeutic target. Owing to its importance in lipogenesis, upregulation of human type I FAS expression has been observed in numerous cancers. Type I FAS is also regarded as important target in antiobesity treatment. Both multidomain enzyme classes - FASs and PKSs - show high structural and functional similarities. Particularly animal type I FAS is most relevant as evolutionary precursor of the PKS family. Therefore, the well characterized FASs are suitable model proteins for the poorly characterized PKSs, to gain deeper understanding in these megasynthases.
Furthermore, fatty acids are considered to be strategically important platform chemicals accessible through sustainable microbial approaches. The recently acquired structural information on FASs provides an excellent understanding of the molecular basis of fatty acid synthesis. The specific understanding of chain-length control, the characterization of a multitude of substrate-specific thioesterases, and the emerging tools and means for metabolic engineering have fostered targeted approaches for modulating chain length. There is large interest in short-chain fatty acids, since these compounds are biotechnologically valuable platform chemicals and biofuel precursors, and attempts on the synthesis of short-chain fatty acids have been reported during the last years.
Primary focus of this thesis lies on the animal type I FASs, which exhibit large conformational variety, as seen in electron microscopy and high-speed atomic force microscopy. Conformational dynamics facilitate productive protein-protein interactions between catalytic domains within the enzyme and aid acyl carrier protein (ACP)-mediated substrate shuttling during the catalytic cycle of fatty acid biosynthesis. To gain deeper insight into the fundamental processes of ACP-mediated substrate shuttling and the underlying conformational dynamics, spectroscopic methods like Förster resonance energy transfer and electron paramagnetic resonance spectroscopy shall be employed. These spectroscopic methods demand site-specific labeling of proteins with fluorophore or spin labels, which can be accomplished with the amber codon suppression technology. Through amber codon suppression, a non-canonical amino acid (ncAA) with an orthogonal functional group is incorporated site-specifically into the protein sequence, which can be used in chemoselective reactions for protein labeling.
This thesis is at the forefront of employing the technology of amber codon suppression for addressing complex biological questions on megasynthases. The successful production of ncAA-modified FASs is challenging. With the aim of incorporating ncAAs into the multidomain 540 kDa large murine FAS, we by far exceed boundaries of documented application of amber codon suppression. Most of the proteins that are reported by Liu & Schultz in applications of amber codon suppression are in the range of 30kDa - for example the TE domain of human FAS. In the same review, the largest protein amber codon suppression was applied to is a potassium channel with roughly 80 kDa. Thus, to the best of my knowledge no protein exceeding 100 kDa has been used in amber codon suppression so far.
In this thesis a low-complex, well-plate based reporter assay is presented, based on an ACP-GFP fusion protein for fast and efficient screening of ncAA incorporation. Reliability and applicability of the reporter assay is demonstrated by successful upscaling to larger protein constructs and increased expression scale.
As outlined in this thesis, we have carefully set up methods for the modification of murine FAS and made several achievements:
(i) We have created our own toolbox with a multitude of suppressor plasmids and various orthogonal pairs. pACU and pACE plasmids are compatible for fast exchange of cassettes, and cloning procedures are optimized for modification of synthetases by site-directed mutagenesis. (ii) We have organic synthesis of several ncAAs stably running in the lab and synthesis of other ncAAs can be established when required. Therefore, extensive screening at moderate costs is possible. (iii) We have established a reporter assay for screening our own library of vectors for amber codon suppression and for optimizing incorporation of ncAAs. (iv) We successfully incorporated ncAAs into subconstructs and full-length murine FAS, and collected initial promising results for the application of these proteins in spectroscopic methods. Thus, laying the foundation for future studies to address fundamental questions of the ACP-mediated substrate shuttling and other conformational dynamics of these enzymes.
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
RNA research is very important since RNA molecules are involved in various gene regulatory mechanisms as well as pathways of cell physiology and disease development.1 RNAs have evolved from being considered as carriers of genetic information from DNA to proteins, with the three major types of RNA involved in protein synthesis, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).2 In addition to the RNAs involved in protein synthesis numerous regulatory non-coding RNAs (ncRNAs) have been discovered in the transcriptome. The regulatory ncRNAs are classified into small ncRNAs (sncRNAs) with transcripts less than 200 nucleotides (nt) and long non-coding RNAs (lncRNAs) with more than 200 nt.3
LncRNAs represent the most diverse and versatile class of ncRNAs that can regulate cellular functions of chromatin modification, transcription, and post-transcription through multiple mechanisms.4 They are involved in the formation of RNA:protein, RNA:RNA and RNA:DNA complexes as part of their gene regulatory mechanism.4,5 The RNA:DNA interactions can be divided into RNA:DNA heteroduplex formation, also called R-loops, and RNA:DNA:DNA triplex formation. In triplex formation, RNA binds to the major groove of double-stranded DNA through Hoogsteen or reverse Hoogsteen hydrogen bonding, resulting in parallel or anti-parallel triplexes, respectively. In vitro studies have confirmed the formation of RNA:DNA:DNA triplexes.6 However, the extent to which these interactions occur in cells and their effects on cellular function are still not understood, which is why these structures are so exciting to study (Chapter I RNA:DNA:DNA Triplexes).
This cumulative thesis investigates several functional and regulatory important RNAs. The first project involves the improved biochemical and biophysical characterization of RNA:DNA:DNA triplex formation between lncRNAs of interest and their target genes. Triplex formation was confirmed by a series of experiments including electromobility shift assays (EMSA), thermal melting assays, circular dichroism (CD), and liquid state nuclear magnetic resonance (NMR) spectroscopy. The following is a summary of the main findings of these publications.
In research article 5.1, the oxygen-sensitive HIF1α-AS1 was identified as a functionally important triplex-forming lncRNA in human endothelial cells using a combination of bioinformatics techniques, RNA/DNA pulldown, and biophysical experiments. Through RNA:DNA:DNA triplex formation, endogenous HIF1α-AS1 decreases the expression of several genes, including EPH receptor A2 (EPHA2) and adrenomedullin (ADM), by acting as an adaptor for the repressive human silencing hub (HUSH) complex, which has been studied by our collaborators in the groups of Leisegang and Brandes.
2) Triplex formation between HIF1α-AS1 and the target genes EPHA2 and ADM was investigated in biochemical and biophysical studies. The EMSA results indicated that HIF1α-AS1 forms a low mobility RNA:DNA:DNA triplex complex with the EPHA2 DNA target sequence. The CD spectrum of the triplex showed distinct features compared to the EPHA2 DNA duplex and the RNA:DNA heteroduplex. Melting curve analysis revealed a biphasic melting transition for triplexes, with a first melting point corresponding to the dissociation of the RNA strand with melting of the Hoogsteen hydrogen bonds. The second, higher melting temperature corresponds to the melting of stronger Watson-Crick base pairing. Stabilized triplexes were formed using an intramolecular EPHA2 DNA duplex hairpin construct in which both DNA strands were attached to a 5 nucleotide (nt) thymidine linker. This approach allowed improved triplex formation with lower RNA equivalents and higher melting temperatures. By NMR spectroscopy, the triplex characteristic signals were observed in the 1H NMR spectrum, the imino signals in a spectral region between 9 and 12 ppm resulting from the Hoogsteen base pairing. To elucidate the structural and sequence specific Hoogsteen base pairs 2D 1H,1H-NOESY measurements of the EPHA2 DNA duplex and the HIF1α-AS1:EPHA2 triplex were performed. The 1H,1H-NOESY spectrum of the HIF1α-AS1:EPHA2 triplex with a 10-fold excess of RNA was semi-quantitatively analyzed for changes in the DNA duplex spectrum. We discovered, strong and moderate attenuation of cross peak intensities in the imino region of the NOESY spectrum. This attenuation was proposed to result from weakening of Watson-Crick base pairing by Hoogsteen hydrogen bonding induced by RNA binding. The Hoogsteen interactions can be mapped based on the analysis of the cross peak attenuation in the NOESY spectra, which we used to generate a structural model of the RNA:DNA:DNA triplex. These biophysical results support the physiological function of HIF1α as a triplex-forming lncRNA that recruits the HUSH-epigenetic silencing complex to specific target genes such as EPHA2 and ADM, thereby silencing their gene expression through RNA:DNA:DNA triplex formation.
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
This thesis reports on the results obtained by expression photoactivatable adenylyl cyclase from Beggiatoa spp. (bPAC) in cholinergic neurons from Caenorhabditis elegans (C. elegans) and the characterization of the role of a single neuron, RIS, during locomotion in the adult animal.
Pharmacological activation of adenylyl cyclases through Forskolin is known to induce increased neuronal output in diverse model organisms through a protein kinase A (PKA) dependent mechanism. Nevertheless, pharmacological assays are not spatially restricted, do not allow for precise and acute activation nor to cessation of the signal. Thus, an optogenetic approach for was selected trough the expression of photoactivatable adenylyl cyclase from Beggiatoa spp. (bPAC) in cholinergic neurons of Caenorhabditis elegans (C. elegans). This model organism was chosen due to its transparency, ease of maintenance, fast generation cycles as well as for being an eutelic animal. Further, its genome has been fully sequenced and the connectome of the neuronal network is known, thus allowing for precise analysis of neuronal function. Furthermore, the molecular mechanisms governing neuronal functions are well conserved up to primates. Mainly two optogenetical tools were applied, bPAC and the light gated cation channel channelrhodopsin 2 (ChR2).
Behavioral assays of bPAC photostimulation in cholinergic neurons recapitulated previous work performed with the photoactivatable adenylyl cyclase from Euglena gracilis (EuPACa), in which swimming frequency and speed on solid substrate were increased. Electrophysiological recordings of body wall muscle (BWM) cells by Dr. Jana F. Liewald showed that bPAC photoactivation led to an increase in miniature postsynaptic current (mPSC) rate and, in contrast to ChR2 invoked depolarization, also amplitude. Analysis of mutants deficient in neuropeptidergic signaling (UNC- 31) via electrophysiology performed by Dr. Jana F. Liewald showed that the increase in mPSC amplitude due to bPAC photoactivation requires neuropeptide release. This was confirmed by co-expression of bPAC with the neuropeptide marker NLP-21::Venus and subsequent fluorescence analysis of release, exploiting the fact that released neuropeptides are ultimately degraded by scavenger cells (coelomocytes). These were enriched with NLP-21::Venus after bPAC photostimulation, but no fluorescence could be observed in the UNC-31 mutants.
Additional analysis of the electrophysiological data performed by myself showed no modulation of mPSC kinetics dues to neuropeptidergic release induced by bPAC. Hence, neuropeptide release and action sites were in the cholinergic neurons, the latter including cholinergic motoneurons.
Dr. Szi-chieh Yu provided electron microscopy images of high pressure frozen, bPAC or ChR2 expressing animals. These were tagged by myself for automatic analysis of ultrastructural properties of the cholinergic presynapse, also during photoactivation of both optogenetic tools. Photoactivation of both induced a reduction of synaptic vesicles, with ChR2 showing a more severe effect. In contrast to ChR2, though, bPAC also reduced the amount of dense core vesicles (DCV), the neuropeptide transporters. Additionally, long bPAC photoactivation as well as ChR2 photoactivation led to the appearance of large vesicles (LV), presumably in response to the increased SV fusion rate. bPAC photostimulation also induced an increase in SV size, not observed after ChR2 photostimulation. In UNC-31 mutants, bPAC photostimulation could not lead to the SV size increase, a further argument for the presynaptic effect of the released neuropeptide. Additional analysis of electrophysiology paired with pharmacology, performed by Dr. Jana F. Liewald, showed that mPSC amplitude increase requires the function of the vesicular acetylcholine transporter.
A further effect observed in the ultrastructure of bPAC photostimulated cholinergic presynapses was a shift in the distribution of SV regarding the dense projection. An analysis of cAMP pathway mutants showed that synapsin is required for bPAC induced behavior effects. Synapsin is known to mediate SV tethering to the cytoskeleton. Here, I show evidence for a new role of synapsin in controlling the availability of DCVs for fusion and thus, in neuropeptidergic signaling.
In the second part of my thesis I characterized the function of the GABAergic interneuron RIS in the neuronal network of C. elegans. RIS was shown to induce lethargus, a sleep-like state, during all larval molts, but its function in the adult animal was not yet described. Specific RIS expression of ChR2 achieved by a recombinase based system allowed to acutely depolarize the neuron during locomotion, which led to an acute behavioral stop. Diverse signal transduction pathway mutants were analyzed showing that the phenotype was induced by neuropeptidergic signaling. Through mutagenesis followed by whole genome sequencing data analysis as well as analysis of RIS specific RNA sequencing data further narrowed the signal transduction pathway to mediate the locomotion stop behavior. Since the neuropeptide and, to some extent, the neuron are conserved across nematodes, an argument is outlined in favor of the conservation of this sleep-like state.
In addition, since ChR2 could induce neuropeptidergic signaling from RIS, secretion of vesicles is regulated by variable pathways depending on the neuronal identity. Nevertheless, expression of bPAC in RIS allowed to optogenetically increase the probability of short stops, as observed by expression of a calcium sensor (GCaMP) in RIS and analysis of its intrinsic activity in the adult animal.
Non-ribosomal peptide synthetases (NRPSs) are modular biosynthetic megaenzymes producing many important natural products and refer to a specific set of peptides in bacteria’s and fungi’s secondary metabolism. With the actual purpose of providing advantages within their respective ecological niche, the bioactivity of the structurally highly diverse products ranges from, e.g., antibiotic (e.g., vancomycin) to immunosuppressive (e.g., cyclosporin A) to cytostatic (e.g., echinomycin or thiocoralin) activity.
An NRPS module consists of at least three core domains that are essential for the incorporation of specific substrates with the 'multiple carrier thiotemplate mechanism' into a growing peptide chain: an adenylation (A) domain selects and activates a cognate amino acid; a thiolation (T) domain shuffles the activated amino acid and the growing peptide chain, which are attached at its post-translationally 4ʹ-phosphopantetheine (4'-PPant) group, between the active sites; a condensation (C) domain links the upstream and downstream substrates. NRPS synthesis is finished with the transfer of the assembled peptide to the C-terminal chain-terminating domain. Accordingly, the intermediate is either released by hydrolysis as a linear peptide chain or by an intramolecular nucleophilic attack as a cyclic peptide.
The NRPS’s modular character seems to imply straightforward engineering to take advantage of their features but appears to be more challenging. Since the pioneering NRPS engineering approaches focused on the reprogramming and replacement of A domains, several working groups developed advanced methods to perform a complete replacement of subdomains or single or multiple catalytic domains.
The first part of this work focusses parts of the publication with the title 'De novo design and engineering of non-ribosomal peptide synthetases', which follows up assembly line engineering with the development of a new guideline. Thereby, the pseudodimeric V-shaped structure of the C domain is exploited to separate the N-terminal (CDSub) and C-terminal (CASub) subdomains alongside a four-AA-long linker. This results in the creation of self-contained, catalytically active CASub-A-T-CDSub (XUC) building blocks. As an advantage over the previous XU concept, the characteristics (substrate- and stereoselectivity) assigned to the C domain subunits are likewise exchanged, and thus, no longer represent a barrier. Furthermore, with the XUC concept, no important interdomain interfaces are disrupted during the catalytic cycle of NRPS, allow to expect much higher production titers. Moreover, the XUC concept shows a more flexible application within its genus origin of building blocks to create peptide libraries. Additionally, with this concept only 80 different XUC building blocks are needed to cover the entire proteinogenic amino acid spectrum.
The second part of this work addresses the influence of the C domain on activity and specificity of A domains. In a comprehensive analysis, a clear influence of different C domains on the in vitro activation rate and the in vivo substrate spectrum could be observed. Further in situ and in silico characterizations indicate that these influences are neither the result of the respective A domains promiscuity nor the C domain’s proofreading, but due to an 'extended gatekeeping' function of the C domain. This novel term of an 'extended gatekeeping' function describes the very nature of interfaces that C domains can form with an A domain of interest. Therefore, the C-A interface is assumed to have a more significant contribution to a selectivity filter function.
The third part of this work combines the NRPS engineering with phylogenetic/evolutionary perspectives. At first, the C-A interface could be precisely defined and further identified to encode equivalent information corresponding to the complete C-A didomain. Moreover, the comparison of NRPSs topology reveals hints for a co-evolutionary relatedness of the C-A didomain and could be shown to reassemble even after separation. In this regard, based on a designed CAopt.py algorithm, the reassembling-compatibility of hybrid interfaces could be determined by scoring of the co-expressed NRPS hybrids. This algorithm also enables the randomization of the interface sequences, thus, leading to the identification of more functional interface variant, which cause significantly higher peptide production and could even be applied to other native and hybrid interfaces.
Physical Biology is a field of life sciences dealing with the extraction of quantitative data from biophysical or molecular biological experiments with different levels of complexity. Such data are further used as parameters for mathematical models of the biological system. These models allow to predict reactions on external stimuli by describing the relevant molecular interactions and are therefore used for example to generate a deeper comprehension of complex human diseases. An essential technique in biophysical research on human diseases is fluorescence microscopy. This is a constantly developed toolbox comprising a large number of specific labeling strategies, as well as a broad spectrum of fluorescent probes. It is further minimal invasive and therefore suitable for measurements in living cells or organisms. The sensitivity of modern photo-detectors even allows for the detection of a single fluorescent probe with an accuracy of approximately 10 nm.
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The model-prediction was further verified by two color SMLM experiments. In this work the development and application of imaging-systems are described which provide quantitative data with single-molecule resolution for systems biological model approaches with a low degree of abstractness. In the near future, the impact of mathematical models in the research field of complex human diseases will increase. The predictions of these models will be more exact, the more detailed and accurate the input parameters will become. This work gives an impression of how quantitative data obtained by SMLM may serve as input parameters for mathematical models at the single-cell level.
The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications
(2020)
Biological Function of Bacterial Dodecins
In this thesis, the dodecins of Mycobacterium tuberculosis (MtDod), Streptomyces coelicolor (ScDod) and Streptomyces davaonensis (SdDod) were studied. Kinetic measurements of the flavin binding of MtDod revealed that the dodecin binding pocket is filled in two distinct steps, for which a kinetic model then was established and verified by experimental data. The analysis with the two-step model showed that the unique binding pocket of dodecins allows them to bind excessive amounts of flavins, while at low flavin concentrations, flavin is released and only weakly bound. This function of flavin buffering prevents accumulation of free oxidised flavins and therefore helps to keep the redox balance of the cell and prevents potential cell damage caused by excessive free flavins. To further gain insights into the role of bacterial dodecins, the effect of knocking out the dodecin encoding gene in S. davaonensis was analysed. The knockout strain showed increased concentrations of various stress related metabolites, indicating that without dodecin the cellular balance is disrupted, which supports the role of dodecins as a flavin homeostasis factor.
With a self-designed affinity measurement method based on the temperature dependent dissociation of the dodecin:flavin complex, which allowed parallel screening of multiple conditions, it was shown that MtDod, ScDod and SdDod have much higher affinities towards FMN and FAD under acidic conditions. Under these conditions, the three dodecins might function as a FMN storage. M. tuberculosis encounters multiple acidic environments during its infection cycle of humans and can adopt a state of dormancy. During recovery from the dormant state, a flavin storage might be beneficial. For some Streptomyces species it was reported that the formed spores are slightly acidic and therefore ScDod and SdDod could function as flavin storages for the spores. Further details on the flavin binding mechanism of MtDod were revealed by a mutagenesis study, identifying the importance of a histidine residue at the fourth position of the protein sequence for flavin binding, but contrary to expectations, this residue seems only to be partly involved in the pH related affinity shift.
The data, reported in this thesis, demonstrates that bacterial dodecins likely function as flavin homeostasis factors, which allow overall higher flavin pools in the cell without disrupting the cellular balance. Further, the reported acid-dependent increase in binding affinity suggests that under certain conditions bacterial dodecins can also function as a flavin storage system.
Application of the Dodecin of M. tuberculosis
In this thesis, the stability of MtDod, ScDod SdDod and HsDod was analysed to find a suitable dodecin for the use as a carrier/scaffold. Therefore, a method to easily measure the stability of dodecins was designed, which measures the ability of the dodecamer to rebind flavins after a heating phase with stepwise increasing temperatures. Using this assay and testing the stability against detergents by SDS PAGE, showed that the dodecamer of MtDod possesses an excellent stability against a vast array of conditions, like temperatures above 95 °C, low pH and about 2% SDS. By solving the crystal structure of ScDod and SdDod, the latter forming a less stable dodecamer, combined with a mutagenesis study, the importance of a specific salt bridge for dodecamer stability was revealed and might be helpful to find further highly stable dodecins.
In addition to the intrinsic high stability of the MtDod dodecamer, also the robustness of the fold was tested by creating diverse MtDod fusion constructs and producing them in Escherichia coli. Here it was shown that MtDod easily tolerates the attachment of proteins up to 4-times of its own size and that both termini can be modified without affecting the dodecamer noticeably. Further, it was shown that MtDod and many MtDod fusion constructs could be purified in high yields via a protocol based on the removal of E. coli proteins through heat denaturation and subsequent centrifugation. In a case study, by fusing diverse antigens from mostly human proteins to MtDod and using these constructs to produce antibodies in rabbits, it was demonstrated that MtDod is immunogenic and presents the attached antigens to the immune system.
The here reported properties of MtDod and to a lesser degree of other bacterial dodecins, show that bacterial dodecins are a valuable addition to the pool of scaffold and carrier proteins and have great potential as antigen carriers.
This work deals with the characterization of three different type II polyketide synthase systems (PKS II) from the Gram-negative bacteria Xenorhabdus and Photorhabdus.
Particular attention was paid to a biochemically underexplored class of aryl polyene (APE) pigments. Bioinformatic analysis of enzymes involved in the biosynthesis and the in vitro reconstruction proved that the synthesis of APEs involves an unusual fatty acid-like elongation mechanism. Furthermore, the discovery of unexpected protein-protein interactions provided new insights into the multienzyme complex formation of this unusual PKS II system. Through collaboration with the groups from Prof. Michael Groll and junior Prof. Nina Morgner, two protein complexes were structurally solved and several native protein multimerization events were identified and allowed us to suggest a possible protein-interaction network. The results are summarized in publication ‘An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments’ (first author; J. Am. Chem. Soc.).
In addition to in vitro-analysis, in vivo-studies were used to investigate the APE compound produced by X. doucetiae in more detail. The activation of the silent biosynthetic gene cluster (BGC) led to the detection of the APE compound in the homologous host. Further combination of homologous expression and targeted deletions of the APE BGC revealed an APE-lipid-like structure. MS-based analyses and purification of intermediates allowed us to deduce structural building blocks of the APE-lipid, which is composed of an APE structural core, a glucosamine residue and an unusual long-chain fatty acid with unusual conjugated double bonds and a phosphoethanolamine head group. In combination with the above stated in vitro-data, we assumed a plausible biosynthetic mechanism of the APE-lipid. The results are summarized in the section ‘Additional Results: Tracing the Full-length APE’.
The biosynthesis of isopropylstilbene (IPS) has already been well-studied by the Bode laboratory and the group of Prof. Ikuro Abe. Studies with Photorhabdus laumondii TT01 by the Bode group revealed the distributed locations and functions of the genes involved in biosynthesis, which originate from two pathways. Particularly, the Bode group first demonstrated that an unusual ketosynthase/cyclase (StlD) catalyzes the condensation of 5-phenyl-2,4-pentadienoyl-ACP and isovaleryl-beta-ketoacyl-ACP via a Michael addition. Such a pathway for stilbene formation is distinct from those widespread in plants. The Abe group solved the structure and biochemical mechanism of StlD and further investigated the aromatization reaction of the aromatase StlC. However, the generation of the required cinnamoyl-precursor 5-phenyl-2,4-pentadienoyl-ACP as a Michael acceptor for this cyclization reaction remained elusive. In this work, we were able to reconstitute the synthesis of the Michael acceptor in vitro, by the action of enzymes from the fatty acid biosynthesis. With the knowledge about the crucial cross-talk from primary and specialized metabolism, we further determined the minimal endowment for stilbene production in a heterologous host. Here, the discovered AasS enzyme StlB is responsible for the generation of cinnamoyl-ACP and among others, plFabH plays a key role as gatekeeper enzyme for further processing. With this information in hand, we were able to obtain IPS production in E. coli. These results are presented in the manuscript ‘Biosynthesis of the Multifunctional Isopropylstilbene in Photorhabdus laumondii Involves Cross-talk Between Specialized and Primary Metabolism’ (co-first author, manuscript).
The biosynthesis of the orange-to-red-pigmented anthraquinones (AQs) is the best-studied type II PKS system according to preliminary results. While several investigations by Brachmann et al. discovered the BGC and the overall product spectrum of the main AQ-256 and its methylated derivatives, data of Quiqin Zhou (Bode group) performed biochemical in vitro analysis paired with in vivo heterologous expression of the ant-genes antA-I. This led to the identification of shunt products that indicated an AQ-scaffold derived from an octaketide intermediate that gets shortened to a heptaketide by the hydrolase AntI, resulting in the main anthraquinone AQ-256. This PKS-shortening mechanism was further confirmed by the protein crystal structure of AntI by the Groll group (publication, minor contributions, co-author, Chem Sci. ‘Molecular Mechanism of Polyketide Shortening in Anthraquinone Biosynthesis of Photorhabdus luminescens’). Further substrate analysis of the P. luminescens AQ-producer and mutants revealed an inhibitory effect of cinnamic acid against the hydrolase AntI. Cinnamic acid might therefore be involved in regulation of AQ biosynthesis (‘Anthraquinone Production is Influenced by Cinnamic Acid’, first author, manuscript).
Biochemical analysis from Quiqin Zhou with the minimal PKS of the AQ-synthase further revealed the exclusive activation of the AQ-ACP by the PPTase AntB. The PPTase is insoluble alone but gets stabilized by the CoA-ligase, most likely inactive, working as a chaperone. Thus, the minimal PKS endowment to produce the octaketide scaffold compromises, besides the ACP, the KS:CLF heterodimer and the MCAT, the co-occurrence of the PPTase AntB and the CoA-ligase AntG. For the first time, X-ray crystallography depicted a minimal PKS in action, by obtaining the structural data of native complexes from an ACP:KS:CLF, the KS:CLF alone and an ACP:MCAT in their non-active and active forms. It was possible to confirm a KS-bound hexaketide, which was built upon heterologous expression of the KS:CLF. Mutagenesis with amino-acids proposed to be involved in protein-protein interactions in the ACP:KS:CLF complex revealed some interesting protein-interaction sites. Additionally, an induced-fit mechanism of the MCAT with the ACP during the malonylation reaction confirmed a monodirectional transfer reaction (‘Structural Snapshots of the Minimal PKS System Responsible for Octaketide Biosynthesis’ co-author, manuscript under review).
Ziel dieser Arbeit war es zum einen Informationen über den Mechanismus in Dodecinproteinen zu gewinnen, der zu der effizienten Fluoreszenzlöschung von gebundenem Riboflavin und einer deutlichen Verlängerung der Lebendsdauer des Chromophors unter Lichteinwirkung führt. Zum anderen sollte mit Hilfe eines kurzen Modellpeptids, das eine Azobenzoleinheit als Photoschalter in seinem Peptidrückgrat enthielt, erste Schritte der Peptidfaltung untersucht werden.
Die Untersuchungen an Dodecinproteinen konzentrierten sich hauptsächlich auf archaeales Dodecin aus Halobacterium salinarum (HsDod). Eine Besonderheit der Dodecinproteine ist, dass sie im Gegensatz zu anderen Flavinbindeproteinen zwei Flavinmoleküle in jeder ihrer sechs identischen Bindetaschen einbauen können. Kurzzeitspektroskopische Untersuchungen im UV/vis-Spektralbereich zeigen, dass nach Photoanregung eines gebundenen Riboflavinmoleküls nach etwa 10 ps der Ausgangszustand wieder erreicht wird. Weiterhin zeigt das Fehlen der stimulierten Emission in den transienten Daten, dass bereits innerhalb der Zeitauflösung des Experiments, in weniger als 150 fs, der erste angeregte Zustand des Riboflavins entvölkert wird. Dies verhindert unerwünschte Reaktionen des Riboflavins und stellt eine Versorgung der Zelle mit diesem wichtigen Baustein für die Biosynthese von FMN und FAD sicher. Die Ergebnisse zeigen außerdem, dass zwei Spezies mit unterschiedlichen spektralen Signaturen und Lebensdauern an dem Löschungsmechanismus und der Wiedererlangung des Ausgangszustands beteiligt sind. Der Vergleich von HsDod-Proteinen in nicht-deuteriertem und deuteriertem Lösungsmittel sowie die spektrale Signatur der Spezies, die mit einer Zeitkonstante von etwa 800 fs zerfällt deuten an, dass ein Elektronen- sowie ein Protonentransfer Teil des Mechanismus sind. Mit Hilfe von HsDod-Proteinen, bei denen der Asparaginsäurerest unterhalb der Bindetasche, der für das Binden eines wasserkoordinierten Magnesiumions verantwortlich ist, gegen Serin (D41S) oder Glutaminsäure (D41E) ausgetauscht war, konnte gezeigt werden, dass das wasserkoordinierte Magnesiumion nicht relevant für den Löschungsmechanismus ist. Dennoch konnte eine Beteiligung von Wassermolekülen nicht ausgeschlossen werden. Die Beteiligung eines Elektronentransfers von einem Tryptophanrest in der Bindetasche auf das photoangeregte Flavin konnte durch Messungen an Dodecinproteinen mit Tryptophan-Derivaten mit unterschiedlichen Ionisationsenergien bestätigt werden.
Die Spezies, die mit einer Zeitkonstante von etwa 5 ps zerfällt, die ebenfalls zu einer Wiederbesetzung des Ausgangszustands führt, konnte nicht eindeutig identifiziert werden. Die spektrale Signatur des zerfallassoziierten Spektrums könnte neben einer neutralen Tryptophanspezies und einem kationischen Riboflavinradikal auch durch schwingungsangeregte Riboflavinmoleküle verursacht werden.
Eine Beteiligung der Ribitylkette am Mechanismus kann aufgrund der Ergebnisse von HsDod-gebundenem Lumiflavin ausgeschlossen werden. Weiterhin konnte anhand der Ergebnisse für HsDod-gebundenes FAD, das in seiner geschlossenen Konformation gebunden wird, wobei der Adeninrest die zweite Position in der Bindetasche besetzt, eine Beteiligung des zweiten Flavins in der Bindetasche am Löschungsmechanismus sowie ein Beitrag zu den Differenzspektren ausgeschlossen werden. Somit dient die Besetzung einer Bindetasche mit zwei Flavinmolekülen vermutlich lediglich der Maximierung der Flavinbeladung. Nicht eindeutig geklärt werden konnte die Frage, ob es sich um einen sequentiellen oder parallelen Mechanismus handelt.
Neben archaealem wurde auch bakterielles Dodecin mittels transienter UV/vis-Spektroskopie untersucht. Für Dodecin aus Halorhodospira halophila (HhDod) konnte ebenfalls eine sehr schnelle Wiedererlangung des Ausgangszustands nach Photoanregung des gebundenen Riboflavins beobachtet werden. Allerdings spiegeln einige Unterschiede in den transienten Daten die Unterschiede in den Bindetaschen von archaealem und bakteriellem Dodecin wider und geben Hinweise darauf, dass die Funktionen in der Zelle für die Dodecine unterschiedlich sind. Diese Hypothese wird durch verschiedene Cofaktoren, Riboflavin und Lumichrom für HsDod und FMN für HhDod, in vivo unterstützt. Die ermittelten Zeitkonstanten sind für das bakterielle Dodecin etwas länger als für das archaeale und die transienten Daten weisen in den spektralen Signaturen der Differenzsignale sowohl Unterschiede als auch Gemeinsamkeiten auf.
Im zweiten Teil dieser Arbeit wurden erste Schritte der Peptidfaltung mit Hilfe eines wasserlöslichen bizyklischen Modellpeptids, das den Photoschalter 4(4’-Aminomethylphenylazo)benzoesäure (AMPB) enthält, untersucht. Hierfür wurden Kurzzeitspektroskopische Messungen im mittleren infraroten Spektralbereich für den Schaltvorgang von der cis-Form des Azopeptids in die trans-Form durchgeführt. Diese Methode erlaubt es, transiente Konformationsänderungen des Peptidrückgrats zu verfolgen. In der cis-Form kann das Peptid mehrere unterschiedliche Konformationen einnehmen, während der Konformationsraum für die trans-Form deutlich eingeschränkt ist. Nach der Photoanregung im Bereich der n-pi*-Bande der Azobenzoleinheit finden die grundlegenden konformationellen Änderungen innerhalb der ersten 10-20 ps statt. Dies wurde durch polarisationsabhängige Messungen bestätigt.
Auf dieser Zeitskala finden die größten Änderungen in den transienten Differenzspektren statt, die auf Konformationsänderungen sowie Kühlprozesse zurückzuführen sind. Diese Prozesse konnten mit einer Zeitkonstanten von 5 ps zusammengefasst werden. Auf längeren Zeitskalen finden weitere Reorganisationsprozesse statt, die mit einer Zeitkonstante von 300 ps zusammengefasst werden können. Bei maximaler Verzögerungszeit des Experiments (1,8 ns) ist der Gleichgewichtszustand noch nicht erreicht und es finden weitere Prozesse auf längeren Zeitskalen statt. Im Vergleich zu einem ähnlichen bereits untersuchten DMSOlöslichen bizyklischen AMPB-Peptid konnte keine schnellere Dynamik durch den Einsatz von Wasser als Lösemittel festgestellt werden, wie es vorangegangene transiente Experimente im UV/vis-Spektralbereich an wasser- und DMSO-löslichen bizyklischen Azopeptiden angedeutet hatten. Die Ergebnisse der transienten Messungen zeigen gute Übereinstimmungen mit molekulardynamischen Rechnungen. Das so gewonnene Modell von den Prozessen nach der Isomerisierung des Photoschalters erlaubt Einblicke in erste Schritte bei der Faltung von Peptiden in ihrem natürlichen Lösungsmittel Wasser und die Zeitskalen der entsprechenden Prozesse.
This cumulative dissertation examines learning in chemistry laboratories, focusing on the challenges and benefits of problem-based learning (PBL) for novices in the lab. It addresses the lack of consistent understanding about what should be learned in labs and why it's important. The research aims to understand what students learn, how they learn, and how lab learning can be improved.
A central concept in PBL labs is Information Literacy, defined as a sociocultural practice enabling learners to identify and use information sources within a specific context as legitimized by the practice community.
The first publication, Wellhöfer and Lühken (2022a), investigates the relationship between PBL and learner motivation. It identifies factors that can foster students' intrinsic motivation in a PBL lab. Autonomy is found to be a key factor, increasing student motivation and presenting a model of the autonomous scientific process. This model involves four steps: information acquisition, designing and applying experimental procedures, experimental feedback, and autonomous process optimization. The results suggest that intrinsic motivation in PBL labs can be enhanced by enabling students to independently execute these steps.
The second publication, Wellhöfer and Lühken (2022b), examines the information process students undergo during their first PBL lab. Using a sociocultural framework, it explores Information Literacy to understand students' handling of information and their perceptions of the information process. The findings reveal that in PBL labs, developing a practical, applicable experimental procedure is crucial for problem-solving and significantly shapes the information-acquisition process. This process is iterative, influenced by new information, leading to more precise information needs. Students assess information quality based on its usefulness for their problem, implementability (considering cognitive understanding, available equipment, and psychomotor skills), and safety.
Furthermore, the role of privileged knowledge forms in evaluating the quality of text sources is explored. Students viewed non-scientific sources as "poor" and scientific sources as "good," yet used both for information gathering. There were discrepancies between their assessment of source quality and actual use, indicating that perception of source quality doesn't always affect their practical decisions.
The third publication, Wellhöfer, Machleid, and Lühken (2023), investigates students' information practices in the lab, focusing on discourse between novice learners and experienced assistants. It shows that theoretical knowledge isn't sufficient for independent practical action, and students need actionable social information from experienced community members. The results highlight that information literacy in the lab for newcomers to a community of practice has distinctive features, and physical experience and tacit knowledge are crucial for learning the methods and group-specific knowledge of the practice community. The article demonstrates how learning information literacy in a practice community requires a social and physical experience and provides insights on how educators can support this process.
Locomotion, the way animals independently move through space by active muscle contractions, is one of the most apparent animal behaviors. However, in many situations it is more beneficial for animals to actively prevent locomotion, for instance to briefly stop before reorienting with the aim of avoiding predators, or to save energy and recuperate from stress during sleep. The molecular and cellular mechanisms underlying such locomotion inhibition still remain elusive. So, the aim of this study was to utilize the practical genetic model organism Caenorhabditis elegans to efficiently tackle relevant questions on how animals are capable of suppressing locomotion.
Nerve cells, mostly called neurons, are known to control locomotion patterns by activating some and inhibiting other muscle groups in a spatiotemporal manner via local secretion of molecules known as neurotransmitters. This study particularly focuses on whether neuropeptides modulate such neurotransmission to prevent locomotion. Neuropeptides are small protein-like molecules that are secreted by specific neurons and that act in the brain by activating G protein-coupled receptors (GPCRs) expressed in other target neurons. They can act as hormones, neuromodulators or neurotransmitters. DNA sequences coding for neuropeptides and their cognate receptors are similar across diverse species and thus indicate evolutionary conservation of their molecular signaling pathways. This could potentially also imply that regulatory functions of specific neuropeptides are also similar across species and are thus meaningful to unravel more general mechanisms for instance underlying locomotion inhibition.
Specifically, we find that the modulatory interneuron RIS constitutes a dedicated stop neuron of which the activity is sufficient to initiate rapid locomotion arrest in C. elegans while maintaining its body posture. Similar to its known function in larval sleep, RIS requires RFamide neuropeptides encoded by the flp 11 gene for this activity, in addition to GABA. Furthermore, we find that spontaneous calcium activity transients in RIS are compartmentalized and correlated with locomotion stop. These findings illustrate that a single neuron can regulate both stopping and sleeping phenotypes.
Secondly, we show that C. elegans RPamide neuropeptides encoded by nlp-22 and nlp-2 regulate sleep and wakefulness, respectively. We unexpectedly find that these peptides activate gonadotropin-releasing hormone (GnRH)-like receptors dose dependently and we highlight their sequence resemblance to other bilaterian GnRH-like neuropeptides. In addition, we show that these receptors are expressed in distinct subsets of neurons that are associated with motor behavior. Finally, we show that nlp 22 encoded peptides signal through GNNR 6 receptors to regulate larval sleep and that nlp 2 encoded peptides require both GNRR 3 and GNRR 6 receptors to promote wakefulness.
In sum, we find that locomotion inhibition in C. elegans is regulated by multiple, but evolutionary conserved RFamide and GnRH-like RPamide neuropeptidergic signaling pathways.
Mechanism of the MHC I chaperone TAPBPR and its role in promoting UGGT1-mediated quality control
(2022)
Information about the health status of most nucleated cells is provided through peptides presented on major histocompatibility complex I (pMHC I) on the cell surface. T cell receptors of CD8+ T cells constantly monitor these complexes and allow the immune system to detect and eliminate infected or cancerous cells. Antigenic peptides displayed on MHC I are typically derived from the cellular proteome and are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-binding cassette (ABC) transporter associated with antigen processing (TAP), which is part of the peptide-loading complex (PLC). In a process called peptide editing, the MHC I-dedicated chaperone tapasin (Tsn) selects peptides for their ability to form stable complexes with MHC I. While initial peptide loading is catalyzed in the confines of the PLC, the second quality control is mediated by TAPBPR, operating in the peptide-depleted cis-Golgi network. TAPBPR was shown to have a more fine-tuning effect on the presented peptide repertoire rather than initial peptide selection. The fundamental mechanism of peptide editing was illuminated by two crystal structures of TAPBPR in complex with peptide-receptive MHC I. Notably, one of these structures reported a structural element that inserted into the peptidebinding pocket. The so-called scoop loop was assumed to be involved in mediating peptide exchange but the underlying mechanism remained undefined. Additionally, latest results suggested that TAPBPR mediates the interaction of the glucosyltransferase UGGT1 with peptide-receptive MHC. To expand the current knowledge of quality control processes in the antigen presentation pathway, the contribution of the scoop loop in peptide editing and the role of TAPBPR in UGGT1-mediated quality control needs to be elucidated. In the first part of this study, TAPBPR proteins with various loop lengths were designed to scrutinize the contribution of the scoop loop in chaperoning peptidereceptive MHC I. In a light-driven approach, the ability of TAPBPR variants to form stable complexes with peptide-free MHC I was tested. These results demonstrated that in a peptide-depleted environment, the scoop loop is of critical importance for TAPBPR to chaperone intrinsically unstable, peptidereceptive MHC I clients. Moreover, fluorescence polarization-based assays allowed the pursuit of peptide exchange in different, native-like environments. Peptide displacement activities of TAPBPR variants illustrated that catalyzed peptide editing is primarily induced by structural elements outside the scoop loop. In a peptide-depleted environment, the scoop loop occupies the position of the peptide C-terminus and acts as an internal peptide surrogate. By combining complex formation and fluorescence polarization experiments, the scoop loop of TAPBPR was shown to be critically important in stabilizing empty MHC I and functions as an internal peptide selector. In the second part of this study, a novel in-vitro glucosylation assay was established to examine the role of TAPBPR in UGGT1-catalyzed re-glucosylation of TAPBPR-bound MHC I clients. Therefore, a peptide-free MHC I-TAPBPR complex with defined glycan species was designed which served as physiological substrate for UGGT1. By subjecting the recombinantly expressed HLA-A*68:02- TAPBPR complex and UGGT1 proteins to the new in-vitro system, UGGT1 was shown to catalyze the transfer of a glucose residue to the N-linked glycan of TAPBPR-bound Man9GlcNAc2-HLA-A*68:02. Moreover, a high-affinity, photocleavable peptide was applied to dissociate the MHC I-chaperone complex. However, in the absence of TAPBPR, no glucosyltransferase activity was observed. Generation of peptide-free MHC I through UV illumination also showed no activity, and only the addition of TAPBPR could restore UGGT1-mediated reglucosylation of the empty MHC I. Independent of the peptide status of HLAA*68:02, the combination of protein glycoengineering and LC-MS analysis implicated that UGGT1 exclusively acts on TAPBPR-chaperoned HLA-A*68:02. The newly established system provided insights into the function of TAPBPR during UGGT1-catalyzed re-glucosylation activity and quality control of MHC I. Taken together, the scoop loop allows TAPBPR to function as MHC I chaperone through stabilizing peptide-receptive MHC I. In a peptide-depleted environment, the loop structure serves as an internal peptide surrogate and can only be dislodged by a high-affinity peptide. Based on these findings, TAPBPR fulfills a dual function in the second level of quality control. On the one hand, TAPBPR functions as peptide editor, shaping the repertoire of presented peptides. On the other hand, TAPBPR mediates peptide-receptive MHC I clients to the folding sensor UGGT1. Here, TAPBPR is essential to promote UGGT1-catalyzed reglucosylation of the N-linked glycan, giving MHC I a second chance to be loaded with an optimal peptide cargo in the peptide loading complex.
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
Polyketides are highly valuable natural products, which are widely used as pharmaceuticals due to their beneficial characteristics, comprising antibacterial, antifungal, immunosuppressive, and antitumor properties, among others. Their biosynthesis is performed by large and complex multiproteins, the polyketide synthases (PKSs). This study solely focuses on the class of type I PKSs, which arrange all their enzymatic domains on one or more polypeptides. Despite their high medical value, little is known about mechanistic details in PKSs.
One central domain is the acyl transferase (AT), which is present in all PKSs and channels small acyl substrates into the enzyme. More precisely, the AT loads the substrates onto the essential acyl carrier protein (ACP), which subsequently shuttles the substrates and all intermediates for condensation and modification to additional domains to build the final polyketide.
Some PKSs use their domains several times during biosynthesis and work iteratively – these are called iterative PKSs. Others feature several sets of domains, each being used only once during biosynthesis – these PKSs are called modular PKSs. All PKSs or PKS modules consist of minimum three essential domains to connect the acyl substrates. Three modifying domains are optional and can enlarge the minimal set. According to the domain composition, the acyl substrate is fully reduced, partly reduced, or not reduced at all. This variation of modifying domains accounts for the huge structural and therefore functional variety of polyketides.
Even though the structure of fatty acids is not exactly reminiscent of polyketides, their biosynthetic pathways are closely related. Fatty acid biosynthesis is carried out by fatty acid synthases (FASs), which share many similarities with PKSs. Both megasynthases feature the same domains, performing the same reactions to connect and modify small acyl substrates. In contrast to PKSs, FASs always contain one full set of modifying domains which is used iteratively, leading to fully reduced fatty acids.
The present thesis extensively analyzes the AT of different PKSs in its substrate selectivity, AT-ACP domain-domain interaction, and enzymatic kinetic properties. The following key findings are revealed through comparison: 1.) ATs of PKSs appear slower than the ones of FASs, which may reflect the different scopes of biosynthetic pathways. Fatty acids as essential compounds in all organisms are needed in high amounts for physiological functions, whereas polyketides as secondary metabolites only require basal concentrations to take effect. 2.) The slower ATs from modular PKSs do not load non-native substrates even in absence of the native substrates. This is different to the faster ATs from iterative PKSs and FASs, which indicates high substrate specificity solely for the ATs from modular PKSs and emphasizes their role as gatekeepers in polyketide synthesis. 3.) The substrate selectivity can emerge in either the first or the second step of the AT-mediated ACP loading and is not assured by a hydrolytic proofreading function.
Moreover, a mutational study on the AT-ACP interaction in the modular PKS 6-deoxyerythronolide B synthase (DEBS) shows that single surface point mutations can influence AT-mediated reactions in a complex manner. Data reveals high enzyme kinetic plasticity of the AT-ACP interaction, which was also recently demonstrated for the interaction in a type II FAS.
Based on these findings, the mammalian FAS is engineered towards a modular PKS-like as- sembly line with the long-term goal to rationally synthesize new products. Basically, three important aspects need to be considered: 1.) AT’s loading needs to be splitted in specific loading of a priming substrate by a priming AT and in specific loading of an elongation substrate by an elongation AT. 2.) FAS-based elongation modules need to be designed with varying domain compositions for introducing functional groups in the product. 3.) Covalent and non-covalent linkers need to be designed for connection of priming and elongation modules.
This study focuses on the first aspect, splitting loading of priming and elongation substrates. An elongation substrate-specific AT is installed in the mammalian FAS via domain swapping. Since ATs from modular PKSs were proven to be substrate specific, these are used to exchange the mammalian FAS AT. This work demonstrates that it is extremely challenging to create stable and functional chimeras, but first essential steps are taken. Proper domain boundaries for AT swapping are established and a stable chimera with 70 % wild type AT activity is created. However, this chimera is only of limited value for application in an elongation module due to the intrinsic slow turnover rate of the wild type AT. Using another PKS AT, a stable elongation module is designed and analyzed in its activity in combination with a priming module. These experiments demonstrate that the loading of priming substrates are successfully suppressed in the elongation module, but nonetheless only minor turnover rates are detected in the assembly line.
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