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Publication Type: search.filters.UBSTyp.DissertationStart date: 2020Institute: search.filters.UBSInstitut.Institut für Biochemie und Technische Biochemie

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Now showing 1 - 10 of 27
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    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.
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    Molecular dynamics simulations of the substrate- and product specificity and mechanism of DNA- and protein lysine methyltransferases
    (2024) Schnee, Philipp; Jeltsch, Albert (Prof. Dr.)
    Protein Lysine Methyltransferases (PKMTs) regulate the epigenetic code of cells and their alteration via somatic mutations are often associated with cancer. The aim of this project is to rationalize the product and substrate specificity of this enzyme family by a combination of biochemical experiments and molecular dynamics simulations. Based on this, a detailed view of the underlying mechanism behind the disease associated mutations shall be gained, which may provide new possibilities for personalized cancer therapies.
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    Allele-specific epigenome editing : development and clinical application
    (2024) Kouroukli, Alexandra; Jeltsch, Albert (Prof. Dr.)
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    Alternative active site confinement by enforcing substrate pre-organization in cyclases
    (2023) Schell, Kristina; Hauer, Bernhard (Prof. Dr.)
    Confinement of an enzyme’s active site is critical to the efficiency of chemical reactions and has been recognized as an important tool for catalysis. Confined active sites facilitate the pre-organization of substrates and intermediates to control the reaction course, protect against premature quenching and provide unique products. The catalytic center activates the substrate, and its activity can be enhanced by residues surrounding the substrate in the active site, changing the local catalyst geometry, and maintaining a constrained structural and/or electronic configuration of the catalytic center. These properties are characteristic of confinement, resulting in the generation of proximity between the substrate and the catalytic center, as well as complementary binding of the substrate into the active site. Effectively, this accelerates the reaction, controls the progress of the reaction, and positions the substrate in a productive conformation. The reaction course is selectively controlled by the stabilization of intermediates and by the interaction of electron-rich residues with electron-poor molecules and vice versa. Despite these benefits, a strongly confined active site is inherently limited to compounds that resemble the native substrate, with only small deviations tolerated. This restricts the applications for new reactivities and prevents broad substrate scopes. In this work, analysis of the enzyme structure in combination with iterative saturation mutagenesis were employed for the development of biocatalysts with alternative confinement and productive substrate pre-organization. To unlock the potential of confined Brønsted acid catalysts this approach was applied on terpene synthases. These enzymes form several carbocations as transition states and intermediates, which can be selectively converted by confinement of the active site. Exploiting the potential of terpene synthases to convert modified terpene scaffolds could provide interesting building blocks with branched isoprene/terpene motifs. In addition, the control of the reaction progression to specific products rather than a mixture of products could be targeted by stabilizing carbocation intermediates or transition states. The present work demonstrates a structure-guided strategy to create an alternative confinement in the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). This strategy aims to create proximity between substrate and catalytic center and complementarity between substrate and active site to obtain productive pre organization of the substrate. This may allow the conversion of geranylacetone (GA), farnesol and farnesylacetone analogs as substrates with modified isoprene patterns. Among different rational and semi-rational approaches only variant G600M (VD1), in which the substrate tunnel was modified, yielded starting activity. Structural analysis of VD1 led to the identification of a bottleneck in the tunnel. This resulted presumably in steric interactions and proximity by decreasing average distances between the double bond of the substrate’s terminal isoprene unit and the catalytic center. Furthermore, a lower fluctuation of these distances around this mean value was observed in VD1 compared to the wild type (WT). These observations support the hypothesis of improved substrate pre-organization and confirm the creation of proximity between the substrate and the catalytic center. The development of a screening method and optimization of reaction conditions facilitated iterative saturation mutagenesis to investigate the evolvability and the potential of the approach. The positions for saturation mutagenesis targeted the shape complementarity of the active site to the GA analog dihydropseudoirone, and the finally developed variant (VD5) showed an 1174-fold increase of the total turnover number and 111-fold increase in catalytic efficiency compared to the WT. Creation of alternative active site confinement demonstrates evolvability and great potential to overcome limitations in the engineering of biocatalysts and allowed the generation of novel building blocks in preparative mg scale. Limitations in the generation of alternative confinement were approached by using lycopene cyclases. These catalysts convert linear lycopene to carotenes under physiological conditions and were mainly studied for the conversion of pseudoionones in this work. The latter substrate could not be converted by AacSHC through generation of alternative confinement. Three different lycopene cyclases were tested, of which CanLCY B showed immediate activity in converting pseudoionone to a monocyclic product. AthLCY-B and AthLCY-E initially showed no conversion of selected terpenes. After optimizing the reaction conditions, a multiple sequence alignment (MSA) was performed to identify non-conserved positions around the catalytic acid of LCY-B. It was hypothesized that these amino acids influence the confinement and the pre-organization of the substrate. Saturation mutagenesis at the identified positions improved β-Ionone formation by 4-fold and conversion by 5% with variant V335L compared to the WT. The applicability of this MSA-based alternative confinement strategy for engineering of further lycopene cyclases was demonstrated by using saturation mutagenesis of the respective residues in AthLCY-E. Under optimized conditions α-Ionone product formation increased 4.5-fold with the best performing variant AthLCY-E S359F compared to WT AthLCY-E. Application of lycopene cyclases to complement activities with challenging steric interactions demonstrated the successful expansion of the diversity for protonation reactions by biocatalysts and the successful application of an MSA-based approach to generate alternative confinement. To further investigate the generation of alternative confinement and expand the toolbox of Brønsted acid catalysis, the acid isomerization of monoterpenes catalyzed by squalene hopene cyclases (SHCs) was investigated. To access selective product formation with monoterpenes a strategy based on cation stabilization was applied to overcome the challenging pre-organization of the cyclic C10 compounds. The focus was on aromatic residues with high electron density and residues surrounding the carbocation to direct the reaction course toward a single monoterpene product. In an initial screening, four monoterpenes were converted by AacSHC, resulting in complex product mixtures. Of these, one monocyclic and one bicyclic substrate were selected for further engineering. The goal here was to increase the formation of terpinen-4-ol, a hydrated monoterpene. In addition, limonene that was not converted by AacSHC was tested. Optimization of reaction conditions and semi-rational engineering to stabilize the carbocation intermediate produced improved variants in terms of selectivity and terpinen-4-ol formation. Saturation mutagenesis of hydrophobic amino acids that interact with the docked substrate and surrounding residues resulted in variants VT3 and VS2, which had the best selectivity and the highest measured terpinene 4-ol formation, respectively. VT3 converted monocyclic terpinolene with a selectivity of 64% and with a 3.4-fold increase in total turnover number (TTN) compared to the WT. The highest terpinene-4-ol formation of 219 µM and a 2-fold increase in TTN compared to the WT was measured with the bicyclic compound sabinene. Features, such as bulkier residues at position 600, found in VT3 and VS2 are likely responsible for generation of alternative confinement by the positioning of aromatic residues, which stabilize cationic intermediates along the reaction trajectory towards terpinene-4-ol formation. Creating alternative confinement shows great potential for overcoming limitations in biocatalyst engineering. Interesting building blocks were generated and new reactivities with improved selectivity in protonation reactions have been discovered. Moreover, this strategy could be used for predicting potential hot spots in enzyme engineering campaigns and data-driven predictions of enzyme functions to decipher the catalytic potential of enzyme scaffolds.
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    EPIC’RISPR: a modular and inducible platform for highly parallel synthetic epigenetics and chromatin imaging in a high-throughput format
    (2021) Oberacker, Phil; Jurkowski, Tomasz P. (Dr.)
    The epigenome describes the sum of epigenetic states in an organism. It consists of biochemical modifications of the DNA and histone proteins, non-coding RNAs and the three-dimensional architecture of the genome. These modifications and structures regulate the genome expression in a cell-type-specific pattern and hence control the development of the whole organism. Research in this field yielded a lot of descriptive information about the correlation between epigenetic marks and gene expression. Unfortunately, we do not know much about the causalities within the epigenetic network. With the discovery of the groundbreaking CRISPR/Cas9 technology, it is now possible to interfere with the epigenetic program. This methodology, which is known as epigenetic editing, allows the recruitment of effector molecules to distinct targets where they introduce or remove specific modifications. By observing the response of the epigenome, we can conclude how the epigenetic network functions. However, this system is somewhat limited regarding the simultaneous modification of multiple loci, which is a necessity for investigating a network. In this thesis, I combined the targeting and recruiting functionality of the CRISPR/Cas9 system in one molecule, the gRNA. Like this, this EPIC’RISPR platform can recruit numerous effector molecules to one or multiple targets simultaneously without interference. I demonstrated this by activating and repressing three target genes with different effector domains at once and by recruiting different fluorophores to several target loci. I further applied this technology to perturb five differently expressed target genes simultaneously with one effector molecule at a time. For this, I performed a large-scale experiment in which I probed the effects of more than 60 epigenetic effector molecules on target gene transcription. I identified several promising candidates which might exhibit synergistic behaviour and hence a stronger and longer-lasting impact on the epigenetic program. Furthermore, I developed ON- and OFF-switches for the EPIC’RISPR system which utilize small molecules to fine-tune the introduced effects arbitrarily. The OFF-switch was further applied for transgene expression control, extending the functionality of this system even further. Additionally, our group developed protocols for the synthesis and functionalisation of paramagnetic beads and their application in the automated high-throughput extraction of nucleic acids. Since its publication, our platform, which we call Bio-On-Magnetic-Beads (BOMB) has since become a hub for collaborations in open-source science, especially during the COVID-19 pandemic.
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    Mechanistic study on the DNA methyltransferase DNMT3A
    (2024) Kunert, Stefan; Jeltsch, Albert (Prof. Dr.)
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    Squalene-Hopene cyclase catalyzed isomerization of monoterpenes
    (2020) Diether, Svenja; Hauer, Bernhard (Prof. Dr.)