Universität Stuttgart
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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 Recent advances in biosurfactant-based association colloids : self-assembly in water(2023) Hellweg, Thomas; Sottmann, Thomas; Oberdisse, JulianRecent studies of self-assembly in binary systems of bio-surfactants, either of microbial origin or saponins extracted from plants, are reviewed. Saponins in water reported in the first section include aescin, glycyrrhizin, and quillaja saponins, while rhamnolipids are discussed in the second section on microbial surfactants. Studies of surface activities are a natural starting point of the characterization of surfactants, but here we focus mainly on physico-chemical and structural properties of self-assembled bulk structures in solution, often characterized by scattering techniques. When quantitative modelling is performed, self-assembly parameters like aggregation numbers, head group areas, and resulting shapes can be followed as a function of physical-chemical parameters like concentration, composition, temperature, or pH. Morphologies include micelles and their structural evolution with addition of other bio- or synthetic surfactants, co-surfactants, proteins or phospholipids.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 Cu-doped calcium phosphate supraparticles for bone tissue regeneration(2024) Höppel, Anika; Bahr, Olivia; Ebert, Regina; Wittmer, Annette; Seidenstuecker, Michael; Carolina Lanzino, M.; Gbureck, Uwe; Dembski, SofiaCalcium phosphate (CaP) minerals have shown great promise as bone replacement materials due to their similarity to the mineral phase of natural bone. In addition to biocompatibility and osseointegration, the prevention of infection is crucial, especially due to the high concern of antibiotic resistance. In this context, a controlled drug release as well as biodegradation are important features which depend on the porosity of CaP. An increase in porosity can be achieved by using nanoparticles (NPs), which can be processed to supraparticles, combining the properties of nano- and micromaterials. In this study, Cu-doped CaP supraparticles were prepared to improve the bone substitute properties while providing antibacterial effects. In this context, a modified sol-gel process was used for the synthesis of CaP NPs, where a Ca/P molar ratio of 1.10 resulted in the formation of crystalline β-tricalcium phosphate (β-TCP) after calcination at 1000 °C. In the next step, CaP NPs with Cu 2+ (0.5-15.0 wt%) were processed into supraparticles by a spray drying method. Cu release experiments of the different Cu-doped CaP supraparticles demonstrated a long-term sustained release over 14 days. The antibacterial properties of the supraparticles were determined against Gram-positive ( Bacillus subtilis and Staphylococcus aureus ) and Gram-negative ( Escherichia coli ) bacteria, where complete antibacterial inhibition was achieved using a Cu concentration of 5.0 wt%. In addition, cell viability assays of the different CaP supraparticles with human telomerase-immortalized mesenchymal stromal cells (hMSC-TERT) exhibited high biocompatibility with particle concentrations of 0.01 mg mL -1 over 72 hours.Item Open Access Association between vitamin D status and eryptosis : results from the German National Cohort study(2023) Ewendt, Franz; Schmitt, Marvin; Kluttig, Alexander; Kühn, Julia; Hirche, Frank; Kraus, Frank B.; Ludwig-Kraus, Beatrice; Mikolajczyk, Rafael; Wätjen, Wim; Bürkner, Paul-Christian; Föller, Michael; Stangl, Gabriele I.Vitamin D, besides its classical effect on mineral homeostasis and bone remodeling, can also modulate apoptosis. A special form of apoptosis termed eryptosis appears in erythrocytes. Eryptosis is characterized by cell shrinkage, membrane blebbing, and cell membrane phospholipid disorganization and associated with diseases such as sepsis, malaria or iron deficiency, and impaired microcirculation. To our knowledge, this is the first study that linked vitamin D with eryptosis in humans. This exploratory cross-sectional trial investigated the association between the vitamin D status assessed by the concentration of plasma 25-hydroxyvitamin D (25(OH)D) and eryptosis. Plasma 25(OH)D was analyzed by LC-MS/MS, and eryptosis was estimated from annexin V-FITC-binding erythrocytes by FACS analysis in 2074 blood samples from participants of the German National Cohort Study. We observed a weak but clear correlation between low vitamin D status and increased eryptosis ( r = − 0.15; 95% CI [− 0.19, − 0.10]). There were no differences in plasma concentrations of 25(OH)D and eryptosis between male and female subjects. This finding raises questions of the importance of vitamin D status for eryptosis in terms of increased risk for anemia or cardiovascular events.Item Open Access Synthesis, in vitro, and computational studies of PTP1B phosphatase inhibitors based on oxovanadium(IV) and dioxovanadium(V) complexes(2022) Kostrzewa, Tomasz; Jończyk, Jakub; Drzeżdżon, Joanna; Jacewicz, Dagmara; Górska-Ponikowska, Magdalena; Kołaczkowski, Marcin; Kuban-Jankowska, AlicjaOne of the main goals of recent bioinorganic chemistry studies has been to design and synthesize novel substances to treat human diseases. The promising compounds are metal-based and metal ion binding components such as vanadium-based compounds. The potential anticancer action of vanadium-based compounds is one of area of investigation in this field. In this study, we present five oxovanadium(IV) and dioxovanadium(V) complexes as potential PTP1B inhibitors with anticancer activity against the MCF-7 breast cancer cell line, the triple negative MDA-MB-231 breast cancer cell line, and the human keratinocyte HaCaT cell line. We observed that all tested compounds were effective inhibitors of PTP1B, which correlates with anticancer activity. [VO(dipic)(dmbipy)]·2 H2O (Compound 4) and [VOO(dipic)](2-phepyH)·H2O (Compound 5) possessed the greatest inhibitory effect, with IC50 185.4 ± 9.8 and 167.2 ± 8.0 nM, respectively. To obtain a better understanding of the relationship between the structure of the examined compounds and their activity, we performed a computer simulation of their binding inside the active site of PTP1B. We observed a stronger binding of complexes containing dipicolinic acid with PTP1B. Based on our simulations, we suggested that the studied complexes exert their activity by stabilizing the WPD-loop in an open position and limiting access to the P-loop.Item Open Access Mechanistic study on the DNA methyltransferase DNMT3A(2024) Kunert, Stefan; Jeltsch, Albert (Prof. Dr.)