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In contrast to its terrestrial counterpart, the metabolic degradation of marine polysaccharides is underexplored. This work aimed to functionally characterize ulvan- and xylan-degrading enzymes from marine Bacteroidetes in order to clarify the metabolic degradation pathway. For the provision of a broad polysaccharide substrate spectrum, ulvan from several different algal sources was extracted to be used in further characterization experiments. The structural differences of these ulvans could be demonstrated by enzymatic degradation with ulvan-active enzymes. In order to clarify the synergistic catalytic effects of polysaccharide sulfatases with GHs in the degradation process of ulvan, several putative sulfatases from F. agariphila were produced recombinantly in E. coli. For that, a coexpression with an FGE encoding gene was required. It could be demonstrated that several glycoside hydrolases are inhibited, if their
substrate is sulfated at the cleavage position and that a previous desulfation using one of the sulfatases enabled the further degradation. Some of the sulfatases showed an endolytic or exolytic cleavage behavior like reported for several GHs. With the combined catalytic activities, it was possible to successfully elucidate the complex ulvan degradation mechanism for the first time, which enables the use of ulvan as a biotechnological source for the production of fine chemicals and pharmaceuticals. This degradation mechanism was shown to be complemented by an alternative pathway that helps with the degradation of uronic acid-containing oligosaccharides. Here, the synergistic effects of a multimodular enzyme containing a sulfatase and rhamnosidase domain were demonstrated. Furthermore, the first dehydratase participating in the degradation of oligosaccharides was revealed. The functional characterization of putative xylan-targeting PULs from two Flavobacteriia revealed the existence of marine endolytic and exolytic xylanases. The enzymes of these PULs were produced recombinantly in E. coli and were used in biocatalysis reactions on xylan from beechwood, xylan from P. palmata or commercial xylooligosaccharide standards. Further side chain-active GHs were found to exclusively be active on either standards or xylan. The great variation of genetic equipment was demonstrated by comparing the enzyme activities of these PUL structures assuming different ecological adaptations of these organisms especially, because these PULs do not code for any putative sulfatases, which is uncommon for PULs targeting xylan. A different degradation behavior of the investigated enzymes suggested a preferred conversion of β-1,4-linked xylan, potentially present in some microalgae. The acquired insight of the metabolic ulvan and xylan utilization greatly expands the scientific knowledge about the ecologic interplays in marine environments concerning the polysaccharide utilization. It indicates the necessity of backup mechanisms for metabolic processes in order to get access to complex marine carbon sources in nature. Several small degradation cascades complement each other to break down substrate compounds to monomeric level for the use of structurally diverse polysaccharides. This expands the insights into the metabolic processes in the degradation of marine polysaccharides, which are an important part of the understanding of the ecological interactions in aquatic habitats and the ocean’s carbon cycle.
The characterization of ulvan- and xylan-active enzymes and the clarification of their substrate scopes allow to use these enzymes in future production of carbohydrate-derived chemical products for many industrial applications, making it possible to use algal waste for recycling to high value materials with even beneficial effect for the environment.
Class I and class II glutaredoxins (Grxs) are glutathione (GSH)-dependent proteins, that function as oxidoreductases (class I) or mediate cellular iron trafficking (class II). Some members of class I Grxs like human Grx2 are able to complex a [2Fe-2S] cluster and form a dimeric holo complex, which renders them catalytically inactive and is the basis for their function as redox sensors. Class II Grxs like human Grx5 also complex [2Fe-2S] clusters, however these proteins transfer the clusters to other proteins. Both functionally distinct classes share a similar thioredoxin fold and conserved interaction sites for the non-covalently binding of GSH, which is required to complex the [2Fe-2S] cluster. Furthermore, the proteins from both classes contain a highly nucleophilic active site cysteine that would allow both classes to catalyze GSH-dependent oxidoreduction reactions. Despite of these similar features, only class I Grxs are able to form a mixed disulfide with GSH and to reversibly transfer it to protein thiols (de-/glutathionylation). Interestingly, neither class I Grxs nor class II Grxs can effectively compensate the loss of an essential member of the other class. Even though some structural differences were described earlier, the basis for their different functions remained unknown. In particular, the lack of catalytic activity of class II Grxs as oxidoreductases could not be explained. Here, we demonstrate that the different conformations of a conserved lysyl side chain are the molecular determinant of the oxidoreductase or Fe-S transfer activity of class I and II Grxs, respectively. A specific loop structure that is conserved in all class II Grxs determines one lysyl conformation that prevents the formation of a mixed disulfide of the active site cysteinyl thiol with GSH. Using engineered mutants of hGrx2 and hGrx5, we demonstrated that the exchange of the distinct loop between the classes results in a loss of oxidoreductase function of class I hGrx2 and the gain of oxidoreductase activity of class II hGrx5. The altered GSH binding mode also profoundly changes the [2Fe-2S] cluster binding of the engineered mutants and thereby also influences stability of the holo complexes, a pre-determinant for [Fe-S] cluster transfer activity. With the minor shift of 2 Å in a conserved lysyl side chain orientation we were not only able to modify the catalytic activity of two small human mitochondrial proteins, but on a much larger scale also provided evidence for the previously unknown structural basis that determines the function of all class I and class II Grxs.
The oxidoreductase activity of hGrx2 was also analyzed in vivo in a model of doxorubicin cell toxicity. Applying a mass spectrometrical approach, we identified various mitochondrial proteins as targets for redox regulation. Furthermore, our results gave reason to reconsider some common assumptions regarding doxorubicin-induced apoptosis and the protective function of mitochondrial Grx2.
Using validated analytical tools and optimized sampling procedures, it was possible to detect a vast number of metabolites from the extracellular space but also from the cytosol of B. subtilis. The results indicate that the complement of the analytical methods was suitable in the monitoring of the metabolome since it allowed a great coverage of physicochemical diverse metabolites. However, a wide number of unknown metabolites/features were also detected. Although broad databases exist that can help in the annotation of metabolites, further investigation is needed in their identification. In unpredictable changing conditions, bacterial cells possess appropriate adaptation strategies for a successful bacterial growth. These rely on sensing mechanisms that globally adjust gene expression via transcription and feedback regulations. The metabolic sensing mechanisms have emerged as key roles in the nutritional information and regulation of cell cycle processes. In this work, a new quality of information regarding the metabolism and adaptation to the absence of key signal mechanisms in B. subtilis was provided. Investigations of cells lacking Pyk uncovered alterations in the import of glucose and pyruvate from the nutritional media. These results gives insights to the pyruvate homeostasis mechanism but also brought new questions concerning the regulation of the CCR. Pyruvate wasn't susceptible to the glucose dependent CCR in Δpyk. The earlier in ux of pyruvate in these cells is in accordance to the newly discovered pyruvate transport mechanism. Also, it was speculated that the lower consumption of external glucose could be a consequence of the impairment of the PTS system in the mutant cells due to the accumulation of glycolytic metabolite FBP. In future studies, insights of the PTS system mechanism should be done in these conditions, that could comprise the determination of HPr phosphorylation and the HPrK activity. This study also arose new questions that should be address, that include the higher secretion of acetoin and 2,3-butanediol, and the lower accumulation of shikimate 3-phosphate by the mutant cells. In an untargeted metabolomic analysis, a vast number of altered features were suggested to be fatty acids metabolites, precursors of phospholipids and LTA. Complementary approaches should be done for the confirmation of these metabolites and the inspection of possible alterations in the membrane structure. In the study of LTA mutants, the accumulation of PG precursors provided a hint of altered cell wall assembly. Although by uorescence microscopy no clear changes were detected, the metabolic results emphasized the previous assumption of the affected hydrolytic activity occurring in the PG. For comprehensive knowledge of the cell wall it would be important to detect and identify more metabolites of the LTA anchor using optimized cromatographic method. These results could be complemented with other omics data sets studies which would help in the elucidation of these key regulatory systems mechanisms.
Blood platelets are primary major players in the coagulation cascade, that act upon damage in blood vessels at the subendothelial surface. During this process, platelets change their shape, release granules and aggregate by cross-linking of integrin αIIbβ3 via fibrinogen. The heterodimeric transmembrane receptor integrin αIIbβ3 is highly expressed on platelets and its regulation is bidirectional. Inside-out signaling leads to increased affinity for ligands due to dramatic rearrangements in the integrin conformation changing from an inactive bent conformation to an extended, high-affinity conformation. The swing-out motion of the integrin head domain enables binding of ligands, e.g. fibrinogen, resulting in outside-in signaling guiding kinase activation, shape change, platelet aggregation and spreading, subsequently.
Agonists (e.g. thrombin) and other triggers (e.g. shear stress) promote the activity of platelets, making the study of specific proteins delicate. Therefore, this PhD thesis describes a biomimetic system used to study αIIbβ3 membrane receptors. Integrin αIIbβ3 was successfully reconstituted into liposomes and characterized by biophysical and molecular biological methods (e.g. dynamic light scattering, transmission electron microscopy, circular dichroism spectroscopy and flow cytometry). The fusion of liposomes to a solid substrate allows the analysis of potential activation triggers and interaction partners concerning their role in integrin αIIbβ3 activation in a lipid bilayer. Among others, quartz-crystal microbalance measurements show that divalent ions and clinically relevant drugs (e.g. unfractionated heparin and quinine), known to be involved in immune thrombocytopenia (ITP), are certainly candidates which induce integrin activation and minor changes in protein secondary structure. In addition, protein corona formation during contact of nanoparticles with blood components, such as fibrinogen, as well as their interaction with artificial platelet model membranes containing integrins were studied. Moreover, lipid environment can be strongly controlled as integrin activation is dependent on the ratio of liquid-ordered and disordered phases within the membrane. Eventually, by exclusion of disturbances of complex external and internal factors, the established system enables the interaction analysis of various substances with receptors under physiological conditions. In contrast, these disturbances are required to understand the complex machinery of cellular processes in vivo. Hence, an expression platform, on the basis of HEK293 cells, was established to study not only the interaction of integrin αIIbβ3 with cytoskeletal networks, but also the impact of mutations on integrin resulting in a disease-like phenotype. Mutations known to induce Glanzmann thrombasthenia (GT) symptoms, were introduced and led to different mechanical properties of integrin-expressing cells, especially during cell adhesion cells. Thereby, generation of biological and medically-relevant processes combined with the biophysical setup contribute to understand disease mechanisms as well as the action of therapeutic agents in diseases such as GT and ITP.
Chiral amines represent high-value fine chemicals serving as key intermediate products in pharmaceutical, chemical and agrochemical industries. In the past decades, application of amine transaminases (ATAs) for stereoselective amination of prochiral ketones emerged to an environmentally benign and economically attractive alternative to transition metal-catalyzed asymmetric synthesis to afford optically pure amines at industrial scale. However, the restricted substrate scope of wild-type transaminases prohibited the conversion of particularly sterically demanding substrates, making protein engineering indispensable. The following thesis covers elaboration of a novel assay for transaminases (Article I) and identification and development of transaminase variants in order to achieve biocatalytic preparation of a set of pharmaceutically relevant model amines, ideally in optically pure form for both stereoisomers, preferentially using asymmetric synthesis and most preferably using isopropylamine as cost-efficient amine donor co-substrate (Article II-IV). The aforementioned target amines and the corresponding precursor ketones (see Scheme 4.1) were conceived and provided by the company F. Hoffmann-La Roche to attain suitable biocatalysts for a variety of potential intermediates for active pharmaceutical ingredients. Protein engineering of the transaminase scaffolds investigated in this thesis comprised: Initial screening for suitable starting enzyme scaffolds, structure-guided rational design of these scaffolds to enable bulky planar substrate acceptance, elaboration of a sequence motif, verification of the motif and preparative-scale asymmetric synthesis reactions (Article II). For non-planar and structurally different target substrates, namely spatially bulky or bi-cyclic bridged substrates, the transaminase variants were specifically refined and a different evolutionary route had to be pursued (Article III and Article IV). These results (Article II) represent not only the first successful endeavor to engineer a PLP-fold type I amine transaminase (commonly denoted as (S)-selective) for the conversion of highly sterically demanding substrates, but also generally expanded the scope of available fold type I amine transaminases by enzymes having a novel and exceptionally broad substrate spectrum. Aside from structure-guided rational protein engineering, as well non-rational methods, such as site-specific saturation mutagenesis or directed evolution, were applied for protein-engineering. In order to do so for all of the target compounds, a novel high-throughput solid phase activity assay for transaminases that was actually developed during the master thesis, was refined and published (Article I). In the context of this thesis, the same assay principle was as well adapted for quantification of specific activities in liquid phase (Article III). A comparison of different methodologies for developing agar plate assays and a detailed step by step protocol of our transaminase assay are illustrated in a book chapter.