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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 Active-site loop variations adjust activity and selectivity of the cumene dioxygenase(2021) Heinemann, Peter M.; Armbruster, Daniel; Hauer, BernhardActive-site loops play essential roles in various catalytically important enzyme properties like activity, selectivity, and substrate scope. However, their high flexibility and diversity makes them challenging to incorporate into rational enzyme engineering strategies. Here, we report the engineering of hot-spots in loops of the cumene dioxygenase from Pseudomonas fluorescens IP01 with high impact on activity, regio- and enantioselectivity. Libraries based on alanine scan, sequence alignments, and deletions along with a novel insertion approach result in up to 16-fold increases in activity and the formation of novel products and enantiomers. CAVER analysis suggests possible increases in the active pocket volume and formation of new active-site tunnels, suggesting additional degrees of freedom of the substrate in the pocket. The combination of identified hot-spots with the Linker In Loop Insertion approach proves to be a valuable addition to future loop engineering approaches for enhanced biocatalysts.Item Open Access Structure, activity and function of the Suv39h1 and Suv39h2 protein lysine methyltransferases(2021) Weirich, Sara; Khella, Mina S.; Jeltsch, AlbertSUV39H1 and SUV39H2 were the first protein lysine methyltransferases that were identified more than 20 years ago. Both enzymes introduce di- and trimethylation at histone H3 lysine 9 (H3K9) and have important roles in the maintenance of heterochromatin and gene repression. They consist of a catalytically active SET domain and a chromodomain, which binds H3K9me2/3 and has roles in enzyme targeting and regulation. The heterochromatic targeting of SUV39H enzymes is further enhanced by the interaction with HP1 proteins and repeat-associated RNA. SUV39H1 and SUV39H2 recognize an RKST motif with additional residues on both sides, mainly K4 in the case of SUV39H1 and G12 in the case of SUV39H2. Both SUV39H enzymes methylate different non-histone proteins including RAG2, DOT1L, SET8 and HupB in the case of SUV39H1 and LSD1 in the case of SUV39H2. Both enzymes are expressed in embryonic cells and have broad expression profiles in the adult body. SUV39H1 shows little tissue preference except thymus, while SUV39H2 is more highly expressed in the brain, testis and thymus. Both enzymes are connected to cancer, having oncogenic or tumor-suppressive roles depending on the tumor type. In addition, SUV39H2 has roles in the brain during early neurodevelopment.Item Open Access Structure, activity and function of the NSD3 protein lysine methyltransferase(2021) Rathert, PhilippNSD3 is one of six H3K36-specific lysine methyltransferases in metazoans, and the methylation of H3K36 is associated with active transcription. NSD3 is a member of the nuclear receptor-binding SET domain (NSD) family of histone methyltransferases together with NSD1 and NSD2, which generate mono- and dimethylated lysine on histone H3. NSD3 is mutated and hyperactive in some human cancers, but the biochemical mechanisms underlying such dysregulation are barely understood. In this review, the current knowledge of NSD3 is systematically reviewed. Finally, the molecular and functional characteristics of NSD3 in different tumor types according to the current research are summarized.Item Open Access Visual analysis of large‐scale protein‐ligand interaction data(2021) Schatz, Karsten; Franco‐Moreno, Juan José; Schäfer, Marco; Rose, Alexander S.; Ferrario, Valerio; Pleiss, Jürgen; Vázquez, Pere‐Pau; Ertl, Thomas; Krone, MichaelWhen studying protein‐ligand interactions, many different factors can influence the behaviour of the protein as well as the ligands. Molecular visualisation tools typically concentrate on the movement of single ligand molecules; however, viewing only one molecule can merely provide a hint of the overall behaviour of the system. To tackle this issue, we do not focus on the visualisation of the local actions of individual ligand molecules but on the influence of a protein and their overall movement. Since the simulations required to study these problems can have millions of time steps, our presented system decouples visualisation and data preprocessing: our preprocessing pipeline aggregates the movement of ligand molecules relative to a receptor protein. For data analysis, we present a web‐based visualisation application that combines multiple linked 2D and 3D views that display the previously calculated data The central view, a novel enhanced sequence diagram that shows the calculated values, is linked to a traditional surface visualisation of the protein. This results in an interactive visualisation that is independent of the size of the underlying data, since the memory footprint of the aggregated data for visualisation is constant and very low, even if the raw input consisted of several terabytes.Item Open Access Enzymkatalysierte regioselektive N-Methylierung und N-Alkylierung von Pyrazolen(2021) Bengel, Ludwig L.; Hauer, Bernhard (Prof. Dr.)Item Open Access Loop-Modifikationen als Engineering Strategie zur Optimierung der Cumol Dioxygenase(2021) Heinemann, Peter M.; Hauer, Bernhard (Prof. Dr.)Item Open Access Modifizierte Enzyme ermöglichen die selektive N‐Alkylierung von Pyrazolen unter Verwendung einfacher Halogenalkane(2021) Bengel, Ludwig L.; Aberle, Benjamin; Egler‐Kemmerer, Alexander‐N.; Kienzle, Samuel; Hauer, Bernhard; Hammer, Stephan C.Die selektive Alkylierung von Pyrazolen ist eine Herausforderung in der Chemie und könnte die Synthese wichtiger Moleküle vereinfachen. In dieser Arbeit berichten wir über eine katalysatorgesteuerte Alkylierung von Pyrazolen durch eine cyclische Kaskadenreaktion mit zwei Enzymen. In diesem enzymatischen System nutzt ein promiskuitives Enzym Halogenalkane als Ausgangsstoffe, um nicht-natürliche Analoga des Cosubstrats S-Adenosyl-l-Methionin zu synthetisieren. Ein zweites engineertes Enzym überträgt die Alkylgruppen in einer hochselektiven C-N-Bindungsknüpfung auf das Pyrazol-Substrat. Das Cosubstrat wird regeneriert und nur in katalytischen Mengen eingesetzt. Für das Enzym-Engineering wurde eine computerbasierte Methode verwendet, um eine Mutantenbibliothek in silico zu entwickeln. In einer Runde von Mutagenese und Screening wurde somit eine promiskuitive Methyltransferase in eine kleine Pyrazol-alkylierende Enzymfamilie umgewandelt. Mit diesem bienzymatischen System konnte die Alkylierung von Pyrazolen (Methylierung, Ethylierung, Propylierung) mit bislang unerreichter Regioselektivität (>99 %), Regiodivergenz und in einem ersten Beispiel in präparativem Maßstab gezeigt werden.Item Open Access Peptide und Fusionsproteine für die Biomineralisation von Hydroxylapatit(2021) Henkes, Thorsten Matthias; Hauer, Bernhard (Prof. Dr.)Mittels des sogenannten Phagen Display wurden Peptide identifiziert, welche an Hydroxylapatit binden. Diese Bindemotive wurden in oberflächenaktive Fusionsproteine integriert. Die Bindung der Phagen-präsentierten Peptide, von synthetischen Peptiden und der Fusionsproteine an Hydroxylapatit sowie der Einfluss von Peptiden und Fusionsproteinen auf die Nukleation von Hydroxylapatit wurden untersucht. Ebenso wurden gebildete Präzipitate mittels SEM EDX und TEM charakterisiert. Auf diese Weise wurden Peptidmotive und Fusionsproteine identifiziert, welche die Nukleation von Hydroxylapatit beschleunigen oder verlangsamen können.Item Open Access Studies of the toluene dioxygenase from Pseudomonas putida F1: influence of active-site positions on hydroxylations of mono- and bicyclic aromatics(2021) Wissner, Julian L.; Hauer, Bernhard (Prof. Dr.)Till today, the selective dearomatizing cis-dihydroxylation of aromatics cannot be performed chemically. Hence, it is truly exceptional how nature evolved Rieske non-heme iron dioxygenases (ROs), capable of performing such challenging reactions. These enzymatic multicomponent systems are not only able to catalyze cis-dihydroxylation reactions with outstanding activity, but also with an excellent enantioselectivity (>99% ee) for a broad range of aromatic substrates. In their natural host, ROs are involved in the initial catabolism of aromatic compounds into intermediates of the tricarboxylic acids cycle. In biocatalysis, the oxyfunctionalizing capabilities of ROs can be utilized to synthetize cis-dihydrodiols from mono- and polycyclic arenes. These hydroxylated compounds are valuable synthons, frequently employed in the synthesis of natural products, pharmaceuticals, and fine chemicals. One of the best experimentally characterized ROs reported in literature is toluene dioxygenase (TDO), from Pseudomonas putida F1, which substrate scope comprises over 100 different compounds. Nevertheless, a restraint hampering TDO application as a biocatalyst for the targeted cis-dihydroxylation of aromatics, is the observed decrease in conversion as substrate size increases, resulting in modest product yields. Therefore, fundamental studies revealing the influence of TDO active-site positions, enabling a better conversion of mono- and especially bulky bicyclic aromatics are compulsory. Such need prompted the present thesis, which focused on TDO as model biocatalysts, since it displays remarkable substrate promiscuity and advantageous features for preparative biotransformations. The central task of this work was fostering the biocatalytic capabilities of TDO toward the cis-dihydroxylation of four aromatic model substrates. The first step was the development of an enhanced recombinant TDO system in Escherichia coli (E. coli), for an efficient dioxygenase overexpression. Therefore, the new platform E. coli BW25113 pBAD18-TDO was established, which exhibited outstanding product formation for the bicyclic substrate naphthalene. Nevertheless, in the attempt to apply such platform for the conversion of small monocyclic aromatics, an unforeseen downstream reaction catalyzed by E. coli, dehydrogenating the generated cis-dihydrocatechols to the corresponding catechols, was discovered. By performing a systematic screening of dehydrogenase deficient single knock-out strains from the KEIO collection, the enzyme glycerol dehydrogenase (GldA) from E. coli was identified as the main responsible for such degradation. These findings drove the development of the enhanced platform E. coli BW25113 ΔgldA pBAD18-TDO, allowing the abolishment of the unwanted secondary reaction. Thus, a semi-preparative biotransformation of benzene was performed utilizing the customized TDO platform, resulting in the isolation of 141 mg (31%) of cis-dihydrocatechol as sole product. The system was also tested for the semi-preparative biotransformation of the bicyclic substrate naphthalene, yielding 287 mg (89%) of enantiopure (1R,2S)-1,2-dihydro-1,2-naphthalenediol. Once the TDO platform was established and successfully applied, the next task was to explore the influence of TDO active-site position F366 in naphthalene conversion by generating and investigating a set of nine TDO variants at this position. Strikingly, the single point variant TDOF366V revealed that the enantioselectivity could be switched completely. Furthermore, semi-preparative naphthalene biotransformations with TDOF366V enabled the synthesis of 101 mg (31%) enantioenriched (1S,2R)-1,2-dihydro-1,2-naphthalenediol (90% ee). It is worth to mention that before this study, this enantiomer was never directly generated either chemically or biocatalytically. The next aim was to expand the TDO-based biocatalyst portfolio by generating a semi-rational designed TDO single- and double mutant library. Thus, the library consisting out of 176 variants was tested for the conversion of the bicyclic substrates naphthalene, 1,2,3,4-tetrahydroquinoline, and 2-phenylpyridine in the pursue to enhance product formation and/or chemo-, regio- and enantioselectivity. These studies highlighted that introduced mutations at the active site hot-spot positions M220, A223 and F366, strongly influences chemo-, regio- and enantioselectivity. Moreover, mutations at positions M220 and A223 also exerted substantial effects on product formation during the conversion of bicyclic (hetero)aromatics. In addition, since the TDO mutant library addressed all 14 non-conserved active site amino acids, it was noticeable that the active site is highly tolerant to the introduction of mutations. Additionally, it could be assessed that the combination of the outperforming mutations into the double variant TDOF114H_A223T entirely abolished the formation of the side product quinoline in 1,2,3,4-tetrahydroquinoline biotransformations. This approach enabled in a semi-preparative biotransformation the selective production of 106 mg (71%) of enantioenriched (R)-1,2,3,4-tetrahydroquinoline-4-ol (94% ee). Further, double variant TDOM220A_V309G exhibited an astonishing 15.1-fold higher conversion of the substrate 2-phenyl-pyridine, in comparison to TDO wild type. This enhancement enabled for the first time, the TDO-catalyzed production of 114 mg (60%) enantiopure (1S,2R)-3-(pyridin-2-yl)cyclohexa-3,5-diene-1,2-diol, along with reduced amounts of 6 mg (4%) 2-phenylpyridin-3-ol, as side product. These compelling findings meet the scope of this thesis, in terms of fostering TDO product formation as well as chemo-, regio- and enantioselectivity for bulky bicyclic (hetero) aromatics. Hence, this dissertation highlights the importance of both, the improvement of the recombinant E. coli BW25113 platform, as well as the enhancement of the biocatalyst TDO via enzyme engineering for the generation of valuable cis-dihydroxylated compounds.