03 Fakultät Chemie
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4
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Item Open Access The Bacteroidetes Aequorivita sp. and Kaistella jeonii produce promiscuous esterases with PET-hydrolyzing activity(2022) Zhang, Hongli; Perez-Garcia, Pablo; Dierkes, Robert F.; Applegate, Violetta; Schumacher, Julia; Chibani, Cynthia Maria; Sternagel, Stefanie; Preuss, Lena; Weigert, Sebastian; Schmeisser, Christel; Danso, Dominik; Pleiss, Juergen; Almeida, Alexandre; Höcker, Birte; Hallam, Steven J.; Schmitz, Ruth A.; Smits, Sander H. J.; Chow, Jennifer; Streit, Wolfgang R.Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.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 Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli(2021) Hunold, Andreas; Escobedo-Hinojosa, Wendy; Potoudis, Elsa; Resende, Daniela; Farr, Theresa; Syrén, Per-Olof; Hauer, BernhardPhenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile.Item Open Access EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells(2023) Đorđević, Marija; Stepper, Peter; Feuerstein-Akgoz, Clarissa; Gerhauser, Clarissa; Paunović, Verica; Tolić, Anja; Rajić, Jovana; Dinić, Svetlana; Uskoković, Aleksandra; Grdović, Nevena; Mihailović, Mirjana; Jurkowska, Renata Z.; Jurkowski, Tomasz P.; Jovanović, Jelena Arambašić; Vidaković, MelitaBeta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.Item Open Access The effect of pooling on the detection of the nucleocapsid protein of SARS-CoV-2 with rapid antigen tests(2021) Berking, Tim; Lorenz, Sabrina; Ulrich, Alexander; Greiner, Joachim; Kervio, Eric; Bremer, Jennifer; Wege, Christina; Kleinow, Tatjana; Richert, ClemensThe COVID-19 pandemic puts significant stress on the viral testing capabilities of many countries. Rapid point-of-care (PoC) antigen tests are valuable tools but implementing frequent large scale testing is costly. We have developed an inexpensive device for pooling swabs, extracting specimens, and detecting viral antigens with a commercial lateral flow test for the nucleocapsid protein of SARS-CoV-2 as antigen. The holder of the device can be produced locally through 3D printing. The extraction and the elution can be performed with the entire set-up encapsulated in a transparent bag, minimizing the risk of infection for the operator. With 0.35 mL extraction buffer and six swabs, including a positive control swab, 43 ± 6% (n = 8) of the signal for an individual extraction of a positive control standard was obtained. Image analysis still showed a signal-to-noise ratio of approximately 2:1 at 32-fold dilution of the extract from a single positive control swab. The relative signal from the test line versus the control line was found to scale linearly upon dilution (R2 = 0.98), indicating that other pooling regimes are conceivable. A pilot project involving 14 participants and 18 pooled tests in a laboratory course at our university did not give any false positives, and an individual case study confirmed the ability to detect a SARS-CoV-2 infection with five-fold or six-fold pooling, including one swab from a PCR-confirmed COVID patient. These findings suggest that pooling can make frequent testing more affordable for schools, universities, and similar institutions, without decreasing sensitivity to an unacceptable level.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 Unknown G protein-coupled estrogen receptor correlates with Dkk2 expression and has prognostic impact in ovarian cancer patients(2021) Fraungruber, Patricia; Kaltofen, Till; Heublein, Sabine; Kuhn, Christina; Mayr, Doris; Burges, Alexander; Mahner, Sven; Rathert, Philipp; Jeschke, Udo; Trillsch, FabianWnt pathway modulator Dickkopf 2 (Dkk2) and signaling of the G protein-coupled estrogen receptor (GPER) seem to have essential functions in numerous cancer types. For epithelial ovarian cancer (EOC), it has not been proven if either Dkk2 or the GPER on its own have an independent impact on overall survival (OS). So far, the correlation of both factors and their clinical significance has not systematically been investigated before. Expression levels of Dkk2 were immunohistochemically analyzed in 156 patient samples from different histologic subtypes of EOC applying the immune-reactivity score (IRS). Expression analyses were correlated with clinical and pathological parameters to assess for prognostic relevance. Data analysis was performed using Spearman’s correlations, Kruskal-Wallis-test and Kaplan-Meier estimates. Highest Dkk2 expression of all subtypes was observed in clear cell carcinoma. In addition, Dkk2 expression differed significantly (p<0.001) between low and high grade serous ovarian cancer. A significant correlation of Dkk2 with the cytoplasmic GPER expression was noted (p=0.001) but not for the nuclear estrogen receptor alpha (ERα) or beta (ERβ). Patients exhibiting both, high expression Dkk2 (IRS>4) and GPER (IRS>8), had a significantly better overall survival compared to patients with low expression (61 months vs. 33 months; p=0.024). Dkk2 and GPER expression correlates in EOC and combined expression of both is associated with improved OS. These findings underline the clinical significance of both pathways and indicate a possible prognostic impact as well as a potential for treatment strategies addressing interactions between estrogen and Wnt signaling in ovarian cancer.