04 Fakultät Energie-, Verfahrens- und Biotechnik
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Item Open Access Studien zur biotechnologischen Anwendung und ökologischen Funktion von Pyrrolochinolinchinon(PQQ)-abhängigen Alkoholdehydrogenasen(2021) Wehrmann, Matthias; Hauer, Bernhard (Prof. Dr.)Item Open Access Development of artificial single and double reading domains to analyze chromatin modification patterns(2018) Mauser, Rebekka; Jeltsch, Albert (Prof. Dr.)The unstructured N-terminal tails of histone proteins carry many different post-translational modifications (PTMs), like methylation, acetylation or phosphorylation. These PTMs can alter the chromatin structure, influence the interaction of adjacent nucleosomes and serve as specific binding sites for histone interacting domains. Currently, the investigation of histone tail PTMs is mainly based on antibodies, however concerns about the specificity of these antibodies and reproducibility of data arouse. Therefore, it was one aim of this thesis to develop alternative approaches to histone tail PTM antibodies. Previous studies already showed that histone modification interacting domains (HiMIDs) can replace histone tail antibodies in a highly effective manner. As part of this work, the TAF3 PHD domain was established as new H3K4me3 specific HiMID. In peptide array binding and Far-western blot assays, the domain showed a specific interaction with H3K4me3 modifications. Also in ChIP like experiments (CIDOP: Chromatin Interacting Domain Precipitation) coupled to qPCR and next generation sequencing, the domain showed a similar performance as validated H3K4me3 antibodies. With the proposal of the histone code hypothesis the question was raised if combinations of histone modifications carry specific biological functions. However, so far, the experimental analysis of the co-occurrence of histone modification on the same nucleosome in a genome-wide manner is a challenging task. For this reason, the main aim of this work was to develop double reading domains in which two histone reading domains are fused together with a flexible linker to achieve simultaneously readout of dual histone tail modifications in a single CIDOP experiment. To validate the concept, the Dnmt3a PWWP domain and the MPP8 Chromo domain were fused together and their specific recognitions of H3K36me2/3 and H3K9me3 histone tail modifications were analyzed. Biochemical investigations like peptide arrays, Far-western blot and western blot experiments showed that both domains specifically interact with their targets and preferentially interact with double modified chromatin. Additionally, the preferred interaction with double modified chromatin could be further verified with binding pocket mutants and methyl-lysine analogues. The newly generated double domain was used in chromatin precipitation experiments to identify genome regions where both modifications are present. The genome-wide distribution of the H3K36me2/3-H3K9me3 showed that this combination of histone marks represents a novel bivalent chromatin state, which is associated with weakly transcribed genes and is enriched for binding sites of ZNF274 and SetDB1. Also in this work, mixed peptide arrays were introduced as new screening method for the efficient analysis of double reading domains. The naturally occurring double reading domain of the BPTF protein was used to demonstrate the capability of this new screening tool. BPTF contains a PHD domain, which binds to H3K4me3 and a Bromo domain, which interacts with acetyl groups of the H4 tail. Synergistic binding to both peptides was shown using the newly developed mixed peptide arrays. Additionally, in the course of this work mixed peptide arrays were used to optimize several of the designed double reading domains. Furthermore, some other double reading domains were generated in this work, like PWWP-ATRX, MPP8 Chromo domain-L-double Tudor and CBX7 Chromo domain-L-MPP8 Chromo domain and analyzed for specific dual readout. Also double reading domains with dual specificity for DNA methylation and histone marks were generated. The firstly used methyl-DNA binding domain of the MBD2 protein showed a strong binding, dominating the effect of the HiMIDs. Therefore, the weaker but still specific methyl-DNA binding domain of the MBD1 protein was used. First experiments with this new fusion constructs showed a simultaneously interaction with chromatin which is associated with DNA methylation and histone PTMs. In summary, the studies with double reading domains showed that with this novel method precipitation of double modified chromatin is possible and that the genome-wide investigation of newly studied bivalent chromatin states is feasible. Therefore, this novel approach makes it possible to analyze many different combinations of histone modifications, investigate their influence on chromatin and gain a deeper understanding of the biological role behind histone tail modification patterns.Item Open Access Identifizierung und Charakterisierung von Influenza-A-Virus Fusionsinhibitoren, die aus einem Doppelmyxovirus Hochdurchsatz-Screen hervorgingen(2019) Weißhaar, Marco; Wolf, Dieter H. (Prof. Dr.)Item Open Access Data integration and data mining for the exploration of enzymatic sequence-structure-function relationships(2018) Buchholz, Patrick C. F.; Pleiss, Jürgen (Prof. Dr.)Item Open Access Enzymatische Hydratisierung kurzkettiger Fettsäuren und Alkene(2018) Demming, Rebecca M.; Hauer, Bernhard (Prof. Dr.)Item Open Access 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.Item Open Access Naphthalen Dioxygenase aus Pseudomonas sp. NCIB 9816-4 : systematische Analyse der aktiven Tasche(2017) Halder, Julia M.; Hauer, Bernhard (Prof. Dr.)Die gezielte Oxyfunktionalisierung von Olefinen gehört zu den am meist gesuchten Reaktionen in der Chemie. Insbesondere die Dihydroxylierung und die daraus resultierenden chiralen, vicinalen 1,2-Diole spielen hierbei eine wichtige Rolle. So werden 1,2-Diole sowohl als chirale Liganden und Auxiliare und als chirale Synthons für Pharmabausteine sowie Agrochemikalien eingesetzt. Eine schnelle und effiziente Möglichkeit für die stereoselektive, asymmetrische Sharpless Dihydroxylierung (AD) von C=C-Doppelbindungen ergibt sich aus der Metall-katalysierten Oxyfunktionalisierung mittels Osmium oder anderen Übergangsmetallen. Neben der guten Ausbeute und der hohen Selektivität, stellen jedoch vor allem die Toxizität der Katalysatoren, sowie auch die Überoxidation und Spaltung der generierten cis-Diole Herausforderungen in der Anwendung dar. Rieske Nicht-Häm Dioxygenasen (ROs) sind eine biologische Alternative zur rein chemischen, asymmetrischen Dihydroxylierung. In der Natur sind diese Multikomponentensysteme, bestehend aus einer hexameren Oxygenase, einem Elektronen-Shuttlemolekül und einer Reduktase, für die Dihydroxylierung von aromatischen Motiven verantwortlich und katalysieren den ersten Schritt im Katabolismus von Aromaten. Mit der Entdeckung dieser effizienten Bio-katalysatoren wurde eine umweltfreundliche Alternative zur chemisch katalysierten Sharpless AD entdeckt. Aufgrund der Verfügbarkeit von Kristallstrukturen wurde die Naphthalen Dioxygenase (NDO) aus Pseudomonas sp. NCIB 9816-4 als ein Vertreter der ROs für das semi-rationale Design ausgewählt und Varianten im aktiven Zentrum des Enzyms generiert. Neben der direkten Katalyse am aromatischen Ring, wurde durch Variation der Substituenten auch die allylische Mono- bzw. die cis-Dihydroxylierung von Alkenylresten in aromatischen Molekülen (z. B. α-Methylstyrol, Allylbenzol) und die Katalyse von C=C-Doppelbindungen in nicht-aromatischen, nicht-planaren Molekülen (z. B. R-Limonen) gezeigt. Aufgrund der Vielfältigkeit dieser Enzyme besteht ein gesteigertes Interesse am biotechnologischen Einsatz, um das enorme Potential und die Vielfältigkeit des biokatalytischen Repertoires dieser Katalysatoren ausschöpfen zu können. Des Weiteren erfolgte die nähere Betrachtung der heterologen Herstellung des Biokatalystors in Escherichia coli, wobei sowohl in vitro als auch in vivo Systeme betrachtet wurden. Hierbei stand im Fall der in vitro Untersuchungen das Zusammenspiel der unterschiedlichen Komponenten des Systems, das Reaktionssetup und der Einfluss des Cosolvents im Mittelpunkt. Für das optimierte in vitro System wurden schließlich folgende Parameter definiert: (I) Verhältnis der Komponenten mit 1 μM Oxygenase, 20 μM Ferredoxin und 5 μM Reduktase, (II) das Reaktionssetup mit 2 mM Substrat in Ethanol bei 30 °C für 2 h, und (III) der Anteil des Cosolvents Ethanol mit 5 %(v/v). Ein Alanin-Scan der zwölf first shell Aminosäuren lieferte im in vitro System bereits erste Indizien für relevante Mutagenese-Hotspots mit den Positionen A206, H295, L307, G204 und V260. Im Gegensatz zum in vivo System wurde im in vitro System eine deutlich erniedrigte Aktivität gegenüber den untersuchten substituierten Aromaten detektiert, weshalb auf eine mangelnde Stabilität der Komponenten im in vitro System geschlossen wurde. Im in vivo System wurde zunächst die Optimierung der Expression forciert, wobei das entwickelte Expressions- und Biotransformationsprotokoll zu einer guten Reproduzierbarkeit in Ganzzellansätzen mit Standardabweichungen von unter 5 % geführt hat. Hierzu wurden frisch transformierte Zellen zur Anzucht (37 °C) in TB-Medium verwendet und bei Erreichen einer optischen Dichte von 0,6-0,8 mit 0,1 mM Isopropyl-β-D-thiogalactopyranosid induziert. Nach 20-stündiger Expression bei 25 °C wurden eine Zellsuspension mit 0,1 mM Kaliumphosphatpuffer (pH 7,2) und 20 mM Glucose (0,2 gBFM/mL) für Ganzzellumsätze hergestellt. Die Reaktion wurde durch Zugabe des in Ethanol gelösten Substrates gestartet und nach 20 h bei 30 °C mit der Zugabe von Lösungsmittel gestoppt. Für die in vivo Untersuchung wurde ein semi-rationaler Mutageneseansatz gewählt, indem alle first shell Aminosäuren mit Alanin, Valin und Isoleucin (36 Varianten) ausgetauscht, sowie 25 Doppelvarianten an den Positionen A206, H295 und V260 generiert wurden. Mit dieser Bibliothek erfolgte die Identifizierung von wichtigen Struktur-Funktionsbeziehungen anhand von unterschiedlich substituierten Styrolderivaten und dem Monoterpen R-Limonen. Mit dem Einbringen einer Punktmutation in der aktiven Tasche konnten deutliche Veränderungen in der Reaktions- und Substratspezifität sowie in der Regio- und Stereoselektivität (≥ 90 %) beobachtet werden, wobei die Restaktivität gegenüber dem natürlichen Substrat Naphthalen (bis > 99 %) erhalten blieb. So stellten sich die Position A206, sowie die gegenüberliegenden Positionen H295, F202, F352, V260 und L307 in der planaren, zylinderförmigen aktiven Tasche als maßgeblich für die Steuerung der Aktivität und Selektivität der NDO dar. Generell konnte eine Abnahme der Aktivität mit steigender Substituentengröße und Verzweigungsgrad (Methyl- bis Pentyl- bzw. tert-Butyl-Reste) detektiert werden. Gleichfalls konnte eine Tendenz für ungesättigte Substituenten am Aromaten beobachtet werden, wobei die Aktivität von mono- über gem-di- und trans-di-substituierte Seitenketten abnahm. Bei der Untersuchung von unterschiedlichen Methoxystyrolderivaten konnte eine gesteigerte Spezifität und Stereoselektivität (≥ 95 %ee) beobachtet werden. Neben Hydroxylierungsreaktionen wurden hierbei auch Dealkylierungsreaktionen beobachtet. Die Dihydroxylierung wurde beim Vorliegen einer zum Aromaten konjugierten C=C-Doppelbindung gegenüber der O-Demethylierung bevorzugt. Lag die C=C-Doppelbindung isoliert zum aromatischen System vor, wurde hingegen eine Präferenz für die O-Demethylierung beobachtet. Grundsätzlich hat sich die NDO als einen guten Startpunkt für die biokatalysierte, asymmetrische Dihydroxylierung erwiesen und durch die systematische Analyse der aktiven Tasche konnten essentielle Stellschrauben für die weitere Verbesserung des Katalysator identifiziert werden.Item Open Access Enzymkatalysierte regioselektive N-Methylierung und N-Alkylierung von Pyrazolen(2021) Bengel, Ludwig L.; Hauer, Bernhard (Prof. Dr.)Item Open Access DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation(2022) Dukatz, Michael; Dittrich, Marianna; Stahl, Elias; Adam, Sabrina; De Mendoza, Alex; Bashtrykov, Pavel; Jeltsch, AlbertSpecific DNA methylation at CpG and non-CpG sites is essential for chromatin regulation. The DNA methyltransferase DNMT3A interacts with target sites surrounded by variable DNA sequences with its TRD and RD loops, but the functional necessity of these interactions is unclear. We investigated CpG and non-CpG methylation in randomized sequence context using wildtype DNMT3A and several DNMT3A variants containing mutations at DNA-interacting residues. Our data revealed the flanking sequence of target sites between the -2 and up to the +8 position modulates methylation rates >100-fold. Non-CpG methylation flanking preferences were even stronger and favor C(+1). R836 and N838 in concert mediate recognition of the CpG guanine. R836 changes its conformation in a flanking sequence-dependent manner and either contacts the CpG guanine or the +1/+2 flank, thereby coupling the interaction with both sequence elements. R836 suppresses activity at CNT sites, but supports methylation of CAC substrates, the preferred target for non-CpG methylation of DNMT3A in cells. N838 helps to balance this effect and prevent the preference for C(+1) from becoming too strong . Surprisingly, we found L883 reduces DNMT3A activity despite being highly conserved in evolution. However, mutations at L883 disrupt the DNMT3A-specific DNA-interactions of the RD loop, leading to altered flanking sequence preferences. Similar effects occur after the R882H mutation in cancer cells. Our data reveal that DNMT3A forms flexible and interdependent interaction networks with the CpG guanine and flanking residues that ensures recognition of the CpG and efficient methylation of the cytosine in contexts of variable flanking sequences.