03 Fakultät Chemie
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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 Biochemical analysis of DNA- and protein methyltransferases using recombinant designer nucleosomes(2022) Bröhm, Alexander; Jeltsch, Albert (Prof. Dr.)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 Mechanistic study on the DNA methyltransferase DNMT3A(2024) Kunert, Stefan; Jeltsch, Albert (Prof. Dr.)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 Regioselective hydration of terpenoids using cofactor-independent hydratases(2019) Schmid, Jens; Hauer, Bernhard (Prof. Dr.)Item Open Access Biocatalytic allylic oxidation with bacterial P450 monooxygenases(2023) Bogazkaya, Anna Maria; Hauer, Bernhard (Prof. Dr.)Diese Forschungsarbeit konzentriert sich auf die Identifizierung und Charakterisierung geeigneter Cytochrom P450 Monooxygenasen (CYPs) in Streptoyceten. Durch eine Dünnschichtchromatographie (DC)-Methode wurden zwei vielversprechende Streptomyces-Stämme identifiziert. Aus diesen Stämmen wurden die Enzyme CYP105A1, CYP105B1, CYP105D5 und CYP170A1 kloniert und in E. coli zur weiteren Untersuchung exprimiert. Da die natürlichen Redoxpartner dieser CYPs unbekannt sind, wurde ein heterologes Redoxsystem aus Pseudomonas putida verwendet. Diese Arbeit liefert wichtige Einblicke für die industrielle Anwendung dieser Enzyme in der biokatalytischen Oxidation. Interessanterweise zeigten die Studienergebnisse, dass drei der ausgewählten CYPs (CYP105B1, CYP105D5 und CYP170A1) zyklische Modell Substrate wie α- oder β-Ionon auf eine regioselektive Weise zu Allylalkoholen hydroxylierten. Diese Reaktion erzeugt spezifisch Allylalkohole, was eine hohe Regioselektivität zeigt - das heißt, die Enzyme zielten gezielt auf bestimmte Teile der Moleküle ab, um sie zu oxidieren. Andererseits führte die Oxidation von azyklischen Substraten mit diesen Enzymen zu einer Mischung von regioisomeren Epoxiden. Dies zeigt eine Vielseitigkeit der Enzyme, aber auch eine Variation in der Produktverteilung, abhängig von der Struktur des Ausgangssubstrats. Diese Ergebnisse tragen dazu bei, das Verständnis für die Funktionsweise und die potenzielle Anwendung von CYPs in der Synthese von spezifischen und wertvollen Produkten zu erweitern. Sie unterstreichen das Potenzial von CYPs als leistungsstarke Werkzeuge in der biotechnologischen und pharmazeutischen Industrie, wo selektive und effiziente Oxidationsmethoden dringend benötigt werden. Zur Vereinfachung der Anwendung dieser Ganzzellkatalysatoren in industriellen Prozessen wurde ein innovativer Ansatz zur Immobilisierung dieser Zellen in einer Latex SF091-Schicht erarbeitet, dank der Zusammenarbeit mit Prof. Michael Flickinger von der NC State University, USA. Nach erfolgreicher Immobilisierung der Zellen zeigten die Reaktionen eine bemerkenswerte Produktselektivität: Bei der Oxidation von Nerol mit CYP154E1 wurden 97% 8-Hydroxynerol und nur 3% 2,3Epoxynerol erzeugt.Item Open Access Deep enzymology studies on the mammalian DNA methyltransferases and methylcytosine dioxygenases(2022) Adam, Sabrina; Jeltsch, Albert (Prof. Dr.)Item Open Access Biochemical characterization of protein lysine methyltransferases-regulation, specificity and effect of somatic cancer mutants(2023) Khella, Mina S.; Jeltsch, Albert (Prof. Dr.)Item Open Access O-Methyltransferasen für die chemo- und regioselektive Alkylierung phenolischer Synthesebausteine(2019) Trauzettel, Philipp Otto; Hauer, Bernhard (Prof. Dr.)Die Alkylierung kleiner Synthesebausteine mit unterschiedlich funktionalisierten Resten gehört zu den wichtigsten Reaktionen der organischen Synthese industriell wichtiger Substanzen wie zum Beispiel Pharmazeutika oder Aromastoffe. Die größte Herausforderung ist dabei die chemo , enantio- und regioselektive Modifikation von multifunktionalisierten Strukturen. In der vorliegenden Arbeit wurde das Katalysepotential der O Methyltransferasen PFOMT aus Mesembryanthemum crystallinum, BcOMT2 aus Bacillus cereus und IEMT aus Clarkia breweri untersucht. Mit diesen sollten synthetisch wichtige Kernstrukturen regioselektiv an einzelnen Hydroxygruppen methyliert bzw. durch den Einsatz von Kofaktor-Analoga mit längeren Alkylresten für eine Folgechemie funktionalisiert werden. Aktivitätsmessungen mittels eines substratunabhängigen HPLC-Screeningassays mit Benzyl-, Catechol-, Pyridin-, Naphthol-, Chinolin-, Coumarin-, Flavonoid- und Benzo-phenon-Derivaten zeigten, dass alle drei Enzyme Substrate methylieren, die ein vicinales, dihydroxyliertes Motiv an einem aromatischen Ringsystem besitzen. PFOMT konnte die Substrate dabei am effektivsten umsetzen und toleriert auch zusätzliche Funktionalisierungen am Ringsystem wie z.B. Halogenierungen, Hetero-zyklen oder Nitro- bzw. Aminogruppen. IEMT war zudem in der Lage auch Substrate zu methylieren, bei denen die Hydroxygruppen nicht unmittelbar nebeneinander positioniert sind, wenn auch mit stark verringerter Aktivität. Die anschließende Untersuchung der Regioselektivität der drei Methyltransferasen mit ausgesuchten Substraten zeigte, dass PFOMT eine strikte meta-Regioselektivität aufweist, während BcOMT2 auch zu geringen Anteilen die para-Position methyliert. IEMT hingegen methyliert bevorzugt die para-Hydroxygruppe der getesteten Substrate. Mittels einer fokussierten Mutantenbibliothek von PFOMT konnten Aminosäuren auf beweglichen Loopregionen (Y51 und M199) identifiziert werden, die ausschlaggebend für dessen strikte meta-Selektivität sind. Der Vergleich zu Literaturbekannten O-Methyltransferasen ähnlicher Struktur, jedoch mit einer para-Selektivität, zeigt, dass sich die flexiblen Loops in dieser Enzymklasse stark unterscheiden und damit die Regioselektivität beeinflussen. Zusätzlich hat jedoch auch die Struktur des Substrates einen starken Einfluss auf dessen Positionierung im aktiven Zentrum und folglich auf die Regioselektivität. Durch den Einsatz von Methionin-Analoga und literaturbekannten Varianten der humanen Methionin Adenosyltransferase hMAT2A war es möglich mit denselben Methyltransferasen Ethyl-, Allyl-, Propargyl-, Cyanomethyl-, Ethylazid-, Fluoro-methyl- und Fluoroethyl-Reste auf das Substrat 3,4 Dihydroxyacetophenon zu übertragen. Die Aktivität ist, mit Ausnahme des Allyl- und Ethyl-Analoga, jedoch deutlich geringer als mit dem natürlichen Kofaktor SAM. Durch eine Vergrößerung der aktiven Tasche von PFOMT im Bereich des zu übertragenden Alkylrestes durch rationales Enzymdesign (bump-hole Strategie) konnte für einzelne Analoga die Aktivität um das bis zu Dreifache erhöht werden.