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Item Open Access Physical interactions strengthen chemical gelatin methacryloyl gels(2019) Rebers, Lisa; Granse, Tobias; Tovar, Günter E. M.; Southan, Alexander; Borchers, KirstenChemically cross-linkable gelatin methacryloyl (GM) derivatives are getting increasing attention regarding biomedical applications. Thus, thorough investigations are needed to achieve full understanding and control of the physico-chemical behavior of these promising biomaterials. We previously introduced gelatin methacryloyl acetyl (GMA) derivatives, which can be used to control physical network formation (solution viscosity, sol-gel transition) independently from chemical cross-linking by variation of the methacryloyl-to-acetyl ratio. It is known that temperature dependent physical network formation significantly influences the mechanical properties of chemically cross-linked GM hydrogels. We investigated the temperature sensitivity of GM derivatives with different degrees of modification (GM2, GM10), or similar degrees of modification but different methacryloyl contents (GM10, GM2A8). Rheological analysis showed that the low modified GM2 forms strong physical gels upon cooling while GM10 and GM2A8 form soft or no gels. Yet, compression testing revealed that all photo cross-linked GM(A) hydrogels were stronger if cooling was applied during hydrogel preparation. We suggest that the hydrophobic methacryloyl and acetyl residues disturb triple helix formation with increasing degree of modification, but additionally form hydrophobic structures, which facilitate chemical cross-linking.Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Enzymatic asymmetric dihydroxylation of alkenes(2016) Gally, Christine; Hauer, Bernhard (Prof. Dr.)The introduction of chirality into C=C double bonds is of special interest in organic synthesis. In particular, the catalytic asymmetric dihydroxylation (AD) of alkenes has attracted considerable attention due to the facile transformation of the chiral diol products into valuable derivatives. By chemical means, the metal-catalyzed AD of olefins provides both stereo- and regiospecific cis-diol moieties. Next to their toxicity, however, these metal catalysts can also lead to byproduct formation as a result of oxidative fission. In nature, Rieske non-heme iron oxygenases (ROs) represent promising biocatalysts for this reaction since they are the only enzymes known to catalyze the stereoselective formation of vicinal cis-diols in one step. ROs are key enzymes in the degradation of aromatic hydrocarbons and can target a wide variety of different arenes. Despite their broad substrate scope, limited data is available for the conversion of unnatural substrates by this class of enzymes. To explore their potential for alkene oxidation, three ROs were tested for the oxyfunctionalization of a set of structurally diverse olefins including linear and cyclic arene-substituted alkenes, cycloalkenes as well as several terpenes. Naphthalene- (NDO), benzene- (BDO) and cumene dioxygenases (CDO) from different Pseudomonas strains where selected as they are amongst the RO enzymes that have already been reported to catalyze the oxidation of a small number of olefins. The majority of compounds from the selected substrate panel could be converted by NDO, BDO or CDO and products were either isolated and identified by NMR analysis or using the authentic standards. Dependent on the substrate, allylic monohydroxylation was found in addition to the corresponding diol products, a reaction which is chemically still most reliably achieved by the use of SeO2 in stoichiometric amounts. However, having been evolved for the dihydroxylation of aromatic compounds, wild type ROs displayed low conversions (< 50%) and modest stereoselectivities (≤ 80% ee/de) for several of the tested olefins. To overcome these limitations, changes in the active site topology of RO catalysts were introduced. A single targeted point mutation that was identified based on sequence and structural comparisons with other members of the RO family proved to be sufficient to generate BDO and CDO variants displaying remarkable changes in regio- and stereoselectivity for various substrates. In particular biotransformations with CDO M232A gave excellent stereoselectivities (≥ 95% ee/de) and good activities (> 90%) also for linear alkenes, which have been reported to be challenging substrates for RO-catalyzed oxyfunctionalizations. Site-saturation mutagenesis at position 232 in CDO revealed a correlation between the steric demand of the amino acid side chain and its influence on regio- and/ or stereoselectivities for styrene and indene. While the wild type enzyme almost exclusively catalyzed the dihydroxylation of the aromatic ring, the regioselectivity was shifted with decreasing side chain size to the terminal vinyl group of styrene, yielding up to 96% of the alkene-1,2-diol. For cis-1,2-indandiol formation, enantiocomplementary enzymes could be generated, a fact further highlighting the importance of position 232 for the engineering of ROs. Moreover, site-saturation mutagenesis of additional residues in the substrate binding pocket of CDO (F278, I288, I336 and F378) identified further positions having an influence on selectivity and product formation for alkene oxidation. To proof the applicability of ROs for organic synthesis, semi-preparative scale biotransformations (70 mg) of selected substrates were performed with CDO M232A. Without further optimization of the reaction set-up, products were successfully isolated in > 30% yield. In addition, up-scaling of (R)-limonene hydroxylation to 4 L in a bioreactor with growing cells gave final isolated product titers of 0.4 g L-1 even though substrate volatility and product toxicity diminished the yield. In conclusion, these examples demonstrated that a single point mutation was sufficient to transform CDO wild type into an efficient catalyst, furthermore constituting the first example of the rational engineering of CDO and BDO enzymes for the oxyfunctionalization of a broad range of alkenes.Item Open Access Peptide controlled shaping of biomineralized tin(II) oxide into flower-like particles(2019) Kilper, Stefan; Jahnke, Timotheus; Wiegers, Katharina; Grohe, Vera; Burghard, Zaklina; Bill, Joachim; Rothenstein, DirkThe size and morphology of metal oxide particles have a large impact on the physicochemical properties of these materials, e.g., the aspect ratio of particles affects their catalytic activity. Bioinspired synthesis routes give the opportunity to control precisely the structure and aspect ratio of the metal oxide particles by bioorganic molecules, such as peptides. This study focusses on the identification of tin(II) oxide (tin monoxide, SnO) binding peptides, and their effect on the synthesis of crystalline SnO microstructures. The phage display technique was used to identify the 7-mer peptide SnBP01 (LPPWKLK), which shows a high binding affinity towards crystalline SnO. It was found that the derivatives of the SnBP01 peptide, varying in peptide length and thus in their interaction, significantly affect the aspect ratio and the size dimension of mineralized SnO particles, resulting in flower-like morphology. Furthermore, the important role of the N-terminal leucine residue in the peptide for the strong organic-inorganic interaction was revealed by FTIR investigations. This bioinspired approach shows a facile procedure for the detailed investigation of peptide-to-metal oxide interactions, as well as an easy method for the controlled synthesis of tin(II) oxide particles with different morphologies.Item Open Access Biochemical characterization and identification of novel substrates of protein lysine methyltransferases(2019) Schuhmacher, Maren Kirstin; Jeltsch, Albert (Prof. Dr.)The methylation of lysine side chains is a prevalent post-translational modification (PTM) of proteins, which is introduced by protein lysine methyltransferases (PKMTs). Histone methylation can have different effects on chromatin structure, lysine methylation of non-histone proteins can regulate protein/protein interactions and protein stability. For most PKMTs currently not all methylation sites are known which limits our understanding of the regulatory role of these enzymes in cells. Therefore, it is an important research aim to gain more information about the substrate spectrum of PKMTs. The identification of the substrate specificity of a PKMT is a very important step on the way to identify new PKMT methylation sites. The focus of this study was the analysis of the substrate specificity of different PKMTs by SPOT peptide arrays and based on this on the identification and validation of possible new methylation substrates. The analysis of the substrate specificity of human SUV39H2 revealed significant differences to its human homolog SUV39H1, although both enzymes methylate the same histone substrate (H3K9). SUV39H2 is more stringent than the SUV39H1, which could be demonstrated by the lack of methylation of SUV39H1 non-histone targets by SUV39H2 and by the fact that it was not possible in this study to identify non-histone substrates for SUV39H2. Kinetic studies showed that SUV39H2 prefers the unmethylated H3K9 as substrate. Moreover, it was shown that the N324K mutation of SUV39H2 which leads to a genetic disease in Labrador retrievers causes a change in folding finally leading to the inactivation of the enzyme. It had been reported by another group that the histone variant H2AX is methylated by SUV39H2. However, the sequence of H2AX K134 does not fit to the substrate specificity profile of SUV39H2 determined in the present work. Follow-up in vitro peptide and protein methylation studies indeed showed that H2AX K134 is not methylated by SUV39H2. This indicates that H2AX methylation by SUV39H2 is most probably a wrong assignment of a substrate to a PKMT. Based on already available specificity data for the SUV39H1 PKMT, the SET8 protein was validated as novel substrate in cellular studies. SET8 is a PKMT itself and it could be shown in this thesis that methylation of SET8 at residue K210 by SUV39H1 stimulated the SET8 activity. In humans, there exist different PKMTs, which methylate H3K36. For example, NSD1, NSD2 and SETD2 which were investigated in this thesis. In literature, it was shown that the oncohistone mutation K36M inactivates NSD2 and SETD2. Steady-state methylation kinetics using a peptide substrate and a K36M peptide as inhibitor revealed that NSD1 is inhibited by this histone oncomutation as well. The steady-state inhibition parameters for all enzymes showed a better binding of the PKMTs to the inhibitor peptide than to the substrate, suggesting some mechanistic similarities in target peptide interaction. The SETD2 is a methyltransferase, which is able to introduce trimethylation of H3K36. During this thesis two substrate specificity motifs of SETD2 were determined using peptide array methylation experiments. Additionally, based on the substrate specificity investigations a super-substrate at peptide and protein level was determined. Furthermore, one novel substrate (FBN1) for SETD2 was discovered and validated. The Legionella pneumophila RomA PKMT was shown previously by our collaborators to methylate H3 at K14. Based on the specificity profile of RomA determined in this study it could be shown that this enzyme methylates seven additional human non-histone proteins. Collaborators tested the methylation of one of the non-histone targets (AROS) and could demonstrate its methylation during the infection of human cells with L. pneumophila. The role of these methylation events in the infection process must be studied in future experiments.Item Open Access Studien zum Einsatz von enzymatischen Lewis- und Brønsted-Säuren in Carbonyl-aktivierten organischen Reaktionen(2016) Bischoff, Jennifer N.; Hauer, Bernhard (Prof. Dr.)Item Open Access Loops und Tunnel : unterschätzte Elemente in Enzymen(2020) Heinemann, Peter M.; Rapp, Lea R.; Hauer, BernhardIn enzymes, the active site is the location where substrates are chemically converted. If this site is deeply buried within the protein, substrates must pass not only through the body of the protein via a tunnel, but also flexible, site decorating loops to access the active site. These elements can act as filters that influence on both substrate specificity and activity. Identifying and understanding how they exert such control has been of growing interest over the past several years.Item Open Access Identification and characterization of IgE‐reactive proteins and a new allergen (Cic a 1.01) from chickpea (Cicer arietinum)(2020) Wangorsch, Andrea; Kulkarni, Anuja; Jamin, Annette; Spiric, Jelena; Bräcker, Julia; Brockmeyer, Jens; Mahler, Vera; Blanca‐López, Natalia; Ferrer, Marta; Blanca, Miguel; Torres, Maria; Gomez, Paqui; Bartra, Joan; García‐Moral, Alba; Goikoetxea, María J.; Vieths, Stefan; Toda, Masako; Zoccatelli, Gianni; Scheurer, StephanChickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.Item Open Access Development of a chemoenzymatic (-)-menthol synthesis(2018) Kreß, Nico; Hauer, Bernhard (Prof. Dr.)Biocatalysis is an emergent research area for the development of efficient and sustainable synthesis processes. A crucial milestone for the better applicability of biocatalysts thereby consists of the increasing knowledge of the adaptability of enzymes for distinct synthetic needs like the conversion of specific molecular structures with defined selectivity. In addition, it is equally important to demonstrate that such novel catalysts are combinable among themselves and with established non enzymatic catalysts to enable unexplored synthetic routes. Using the example of the chemoenzymatic synthesis of (-)-menthol from citral, this work therefore addresses the development and applicability of such evolved enzyme catalysts for the synthesis of an industrially relevant molecule. In this complementary synthetic route inspired from an existing industrial process, a mixture of citral isomers is reduced to citronellal using an R-selective ene reductase. In a subsequent Prins reaction, the selective cyclization of R-citronellal to (-)-isopulegol is achieved by the application of an engineered squalene hopene cyclase variant. The final reduction to (-)-menthol proceeds by hydrogenation on a palladium catalyst. Especially the first catalytic step enables an immediate synthetic advantage in comparison to the currently performed industrial process. So far, no catalyst is applied converting both isomers of citral R-selectively at the same time. Both isomers have to be separated under high energy expenditure by distillation prior to reduction. No enzymatic catalyst is described displaying this reactivity yet. As, however, the opposite enantioconvergent S-selective citral reduction by ene reductases is known, the development of an enzyme catalyst constituted an attractive solution for this limitation. Hence, a focus of the work laid on the inversion of the S-selectivity of the citral reduction by NCR ene reductase from Zymomonas mobilis by enzyme engineering. The studies started by characterization of the citral reduction by NCR wild type. Next to the determination of the course of the reaction over time, semi empiric quantum mechanics calculations on the oxidative half reaction of this conversion were carried out. The calculations suggest a so far undescribed catalytic role of an arginine at position 224 for a facilitated hydride transfer and a more complex proton shift involving water molecules in the reaction. The subsequently performed engineering comprised the identification of selectivity determining amino acid positions W66, Y177, I231 and F269 in the active site of the enzyme followed by their variation in an iterative combinatorial fashion. In order to enable the analysis of the multitude of generated enzyme variants, a whole cell screening was developed using chiral gas chromatography. Thereby, the triple variant W66A/I231R/F269V was created converting E/Z-citral in the whole system to R-citronellal with an enantiomeric excess of 89 %. It could be determined that a cell induced citral isomerization leads to increased enantioselectivity in comparison to using purified enzyme. Especially for the influence of the selectivity determining positions W66 and I231 an increased understanding of structure function relations was achieved during the course of semi rational enzyme evolution by the separated analysis of single citral isomers and by supportive in silico analyses like docking and molecular dynamics simulations. The subsequent integration of the established variant A419G/Y420C/G600A of the squalene hopene cyclase from Alicyclobacillus acidocaldarius is remarkable catalyzing the Prins cyclization to (-)-isopulegol with an enantiomeric excess of 99 % and a diastereoselectivity of 90 %. In this context, the enzyme’s underlying Brønsted acid chemistry could be evolved towards the in nature unknown Prins reaction reactivity. In this work it could be shown that enzyme catalysts acquired by such chemical inspection can be implemented in application oriented synthetic routes. In combination with the developed selective ene reductase, the bienzymatic cascade to (-)-isopulegol was successfully performed and characterized. For the final reduction to (-)-menthol an established heterogeneous catalyst like palladium on charcoal could be applied under hydrogen atmosphere. This demonstrates nicely that novel biocatalysts can be combined with approved synthetic processes. With the attained insights, highly valuable (-)-menthol was made accessible for the first time by a chemoenzymatic cascade using an isomeric mixture of citral on preparative scale with 7 % isolated yield. This work not only highlights different strategies for the development of novel biocatalysts, but also contributes to their possible synthetic applicability in the synthesis of industrially relevant molecules.Item Open Access Mechanistic study on the DNA methyltransferase DNMT3A(2019) Emperle, Max; Jeltsch, Albert (Prof. Dr.)The phenotypical and functional diversity of mammalian cell types can be attributed to a large extent to epigenetic signals that determine and stabilize gene expression profiles. One of the most important types of epigenetic signals is DNA methylation. This modification is set early in development by the de novo DNA methyltransferases DNMT3A and DNMT3B, and is found predominantly at the C5 position of cytosine bases in a CpG dinucleotide context. The accurate setting of DNA methylation patterns is critical for normal development and is determined by the precise recruitment and control of DNMT activity on chromatin. In this work, four main directions of research were undertaken, with the ultimate goal of shedding novel mechanistic insights into the mechanism of DNMT3A, its regulation by chromatin signals and interaction partners, as well as the dysregulation of this enzyme in cancer. Furthermore, the potential of DNMT3A to generate 3-methylcytosine as a side reaction was explored. The DNA methyltransferase DNMT3A has been shown to multimerize on DNA and to form large multimeric protein/DNA fibers. However, it has also been postulated that this enzyme can methylate DNA in a processive manner, a property incompatible with fiber formation. By using a dedicated set of biochemical experiments, I was able to show that the DNA methylation rate of DNMT3A increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide, which cannot accommodate DNMT3A polymers. Methylation experiments over a range of enzyme concentrations and with substrates containing one or two CpG sites did not provide evidence for a processive mechanism. The addition of a catalytically inactive DNMT3A mutant was found to increase the DNA methylation rate by DNMT3A on the long substrate but not on the short one. Together, these data clearly indicate that DNMT3A binds to DNA in a cooperative reaction and the formation of protein/DNA fibers increases the DNA methylation rate. These results contribute mechanistic insights into the mode by which DNA methylation patterns are established during development. The second project dealt with characterizing the effects of the R882H exchange on DNMT3A. The R882H mutation is found in the DNA binding interface of DNMT3A and is frequently observed in acute myeloid leukemia (AML). By establishing a double-tag affinity purification system, I was able to show that the mutation only leads to a minor reduction in overall DNA methylation activity in mixed R882H/wildtype DNMT3A complexes. However, a pronounced change in flanking sequence preference of the DNMT3A-R882H mutant was found. Accordingly, a substrate designed to contain the target CpG site flanked by sequences preferred by R882H was better methylated by the variant than by the wildtype enzyme. Together, these data strongly argue against a dominant-negative effect of the R882H mutation and rather propose a site-specific gain-of-activity effect. These findings are in agreement with a recently determined structure of DNMT3A in complex with DNA and they might explain the high prevalence of this specific point mutation in AML. The third project was built on previous data from the lab, documenting a strong and direct interaction between the ADD domain of DNMT3A and the TRD domain of the 5mC reading protein MECP2. These experiments revealed that through its binding, MECP2 allosterically stabilizes the autoinhibitory conformation of DNMT3A, resulting in a strong inhibition of enzymatic activity in vitro. The interaction between these two proteins and its associated inhibition could be disrupted by unmodified histone H3. In my work, I further validated the interaction between the ADD and the TRD domains by size exclusion chromatography. Also, by generating cell lines with stable over-expression of MECP2, I could show that MECP2 inhibits DNMT3A activity in cells. Together, the data from this study offer unprecedented insights into the regulation of DNMT3A by the combined action of chromatin modifications and interaction partners. Accordingly, depending on the modification status of the H3 tail at the target site, MECP2 can act as either a repressor or activator of DNA methylation. The last project dealt with the coevolution between DNA methylation and DNA repair systems, a very exciting topic that was addressed in close collaboration with the laboratory of Dr. Peter Sarkies (MRC London). By performing in vitro methylation experiments with the catalytic domain of DNMT3A, I could show that, in addition to 5mC, DNMT3A can also introduce 3mC, a modification which represents an alkylation damage of DNA. This study provides a new evolutionary perspective on the loss of DNA methylation that is observed in many species.