Universität Stuttgart
Permanent URI for this communityhttps://elib.uni-stuttgart.de/handle/11682/1
Browse
Search Results
Item Open Access Mechanistic studies on the DNA methyltransferases DNMT3A and DNMT3B(2021) Dukatz, Michael; Jeltsch, Albert (Prof. Dr.)In this work, both regulatory and catalytic mechanisms of de novo methyltransferases were investigated, which include interactions with other proteins and the specific recognition of the substrate sequence. Another part of this work strived to elucidate how enzymatic generation of 3-methylcytosine by DNMT3A can occur.Item Open Access Identifying and engineering bottlenecks of autotrophic isobutanol formation in recombinant C. ljungdahlii by systemic analysis(2021) Hermann, Maria; Teleki, Attila; Weitz, Sandra; Niess, Alexander; Freund, Andreas; Bengelsdorf, Frank Robert; Dürre, Peter; Takors, RalfClostridium ljungdahlii (C. ljungdahlii, CLJU) is natively endowed producing acetic acid, 2,3-butandiol, and ethanol consuming gas mixtures of CO2, CO, and H2 (syngas). Here, we present the syngas-based isobutanol formation using C. ljungdahlii harboring the recombinant amplification of the “Ehrlich” pathway that converts intracellular KIV to isobutanol. Autotrophic isobutanol production was studied analyzing two different strains in 3-L gassed and stirred bioreactors. Physiological characterization was thoroughly applied together with metabolic profiling and flux balance analysis. Thereof, KIV and pyruvate supply were identified as key “bottlenecking” precursors limiting preliminary isobutanol formation in CLJU[KAIA] to 0.02 g L-1. Additional blocking of valine synthesis in CLJU[KAIA]:ilvE increased isobutanol production by factor 6.5 finally reaching 0.13 g L-1. Future metabolic engineering should focus on debottlenecking NADPH availability, whereas NADH supply is already equilibrated in the current generation of strains.Item Open Access Structure, activity and function of the NSD3 protein lysine methyltransferase(2021) Rathert, PhilippNSD3 is one of six H3K36-specific lysine methyltransferases in metazoans, and the methylation of H3K36 is associated with active transcription. NSD3 is a member of the nuclear receptor-binding SET domain (NSD) family of histone methyltransferases together with NSD1 and NSD2, which generate mono- and dimethylated lysine on histone H3. NSD3 is mutated and hyperactive in some human cancers, but the biochemical mechanisms underlying such dysregulation are barely understood. In this review, the current knowledge of NSD3 is systematically reviewed. Finally, the molecular and functional characteristics of NSD3 in different tumor types according to the current research are summarized.Item Open Access Visual analysis of large‐scale protein‐ligand interaction data(2021) Schatz, Karsten; Franco‐Moreno, Juan José; Schäfer, Marco; Rose, Alexander S.; Ferrario, Valerio; Pleiss, Jürgen; Vázquez, Pere‐Pau; Ertl, Thomas; Krone, MichaelWhen studying protein‐ligand interactions, many different factors can influence the behaviour of the protein as well as the ligands. Molecular visualisation tools typically concentrate on the movement of single ligand molecules; however, viewing only one molecule can merely provide a hint of the overall behaviour of the system. To tackle this issue, we do not focus on the visualisation of the local actions of individual ligand molecules but on the influence of a protein and their overall movement. Since the simulations required to study these problems can have millions of time steps, our presented system decouples visualisation and data preprocessing: our preprocessing pipeline aggregates the movement of ligand molecules relative to a receptor protein. For data analysis, we present a web‐based visualisation application that combines multiple linked 2D and 3D views that display the previously calculated data The central view, a novel enhanced sequence diagram that shows the calculated values, is linked to a traditional surface visualisation of the protein. This results in an interactive visualisation that is independent of the size of the underlying data, since the memory footprint of the aggregated data for visualisation is constant and very low, even if the raw input consisted of several terabytes.Item Open Access Active-site loop variations adjust activity and selectivity of the cumene dioxygenase(2021) Heinemann, Peter M.; Armbruster, Daniel; Hauer, BernhardActive-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.Item Open Access Structure, activity and function of the Suv39h1 and Suv39h2 protein lysine methyltransferases(2021) Weirich, Sara; Khella, Mina S.; Jeltsch, AlbertSUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment.Item Open Access Physikalische und chemische Wechselwirkungen in Gelatine-Methacryloyl-Lösungen und deren Vernetzung zu Hydrogelen als Trägerstruktur für Gelenkknorpel-Äquivalente(2021) Rebers, Lisa; Tovar, Günter (Prof. Dr.)Item Open Access Peptide und Fusionsproteine für die Biomineralisation von Hydroxylapatit(2021) Henkes, Thorsten Matthias; Hauer, Bernhard (Prof. Dr.)Mittels des sogenannten Phagen Display wurden Peptide identifiziert, welche an Hydroxylapatit binden. Diese Bindemotive wurden in oberflächenaktive Fusionsproteine integriert. Die Bindung der Phagen-präsentierten Peptide, von synthetischen Peptiden und der Fusionsproteine an Hydroxylapatit sowie der Einfluss von Peptiden und Fusionsproteinen auf die Nukleation von Hydroxylapatit wurden untersucht. Ebenso wurden gebildete Präzipitate mittels SEM EDX und TEM charakterisiert. Auf diese Weise wurden Peptidmotive und Fusionsproteine identifiziert, welche die Nukleation von Hydroxylapatit beschleunigen oder verlangsamen können.Item Open Access Modifizierte Enzyme ermöglichen die selektive N‐Alkylierung von Pyrazolen unter Verwendung einfacher Halogenalkane(2021) Bengel, Ludwig L.; Aberle, Benjamin; Egler‐Kemmerer, Alexander‐N.; Kienzle, Samuel; Hauer, Bernhard; Hammer, Stephan C.Die selektive Alkylierung von Pyrazolen ist eine Herausforderung in der Chemie und könnte die Synthese wichtiger Moleküle vereinfachen. In dieser Arbeit berichten wir über eine katalysatorgesteuerte Alkylierung von Pyrazolen durch eine cyclische Kaskadenreaktion mit zwei Enzymen. In diesem enzymatischen System nutzt ein promiskuitives Enzym Halogenalkane als Ausgangsstoffe, um nicht-natürliche Analoga des Cosubstrats S-Adenosyl-l-Methionin zu synthetisieren. Ein zweites engineertes Enzym überträgt die Alkylgruppen in einer hochselektiven C-N-Bindungsknüpfung auf das Pyrazol-Substrat. Das Cosubstrat wird regeneriert und nur in katalytischen Mengen eingesetzt. Für das Enzym-Engineering wurde eine computerbasierte Methode verwendet, um eine Mutantenbibliothek in silico zu entwickeln. In einer Runde von Mutagenese und Screening wurde somit eine promiskuitive Methyltransferase in eine kleine Pyrazol-alkylierende Enzymfamilie umgewandelt. Mit diesem bienzymatischen System konnte die Alkylierung von Pyrazolen (Methylierung, Ethylierung, Propylierung) mit bislang unerreichter Regioselektivität (>99 %), Regiodivergenz und in einem ersten Beispiel in präparativem Maßstab gezeigt werden.Item Open Access Enzymkatalysierte regioselektive N-Methylierung und N-Alkylierung von Pyrazolen(2021) Bengel, Ludwig L.; Hauer, Bernhard (Prof. Dr.)